Recombinant swinepox virus

ABSTRACT

This invention provides a recombinant swinepox virus comprising a foreign DNA sequence which is (a) inserted into the swinepox virus genomic DNA, wherein the foreign DNA sequence is inserted within a region of the genome which corresponds to the 2.0 kb HindIII to BglII subfragment located within the HindIII M fragment of the swinepox virus genome and (b) is expressed in a swinepox virus infected host cell. The invention further provides homology vectors, vaccines and methods of immunization.

This application is a continuation-in-part of international applicationPCT/US94/08277, filed Jul. 22, 1994, which is a continuation-in-part ofU.S. Ser. No. 08/097,554, filed Jul. 22, 1993, now U.S. Pat. No.5,869,312, and U.S. Ser. No. 07/820,154 filed Jan. 13, 1992, now U.S.Pat. No. 5,382,425.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these publications maybe found at the end of the specification immediately preceding theclaims. The disclosures of these publications are hereby incorporated byreference into this application.

BACKGROUND OF THE INVENTION

Swinepox virus (SPV) belongs to the family Poxviridae. Viruses belongingto this group are large, double-stranded DNA viruses thatcharacteristically develop in the cytoplasm of the host cell. SPV is theonly member of the genus Suipoxvirus. Several features distinguish SPVfrom other poxviruses. SPV exhibits species specificity (18) compared toother poxviruses such as vaccinia which exhibit a broad host range. SPVinfection of tissue culture cell lines also differs dramatically fromother poxviruses (24). It has also been demonstrated that SPV does notexhibit antigenic cross-reactivity with vaccinia virus and shows nogross detectable homology at the DNA level with the ortho, lepori, avior entomopox virus groups (24). Accordingly, what is known and describedin the prior art regarding other poxviruses does not pertain a priori toswinepox virus.

SPV is only mildly pathogenic, being characterized by a self-limitinginfection with lesions detected only in the skin and regional lymphnodes. Although the SPV infection is quite limited, pigs which haverecovered from SPV are refractory to challenge with SPV, indicatingdevelopment of active immunity (18).

The present invention concerns the use of SPV as a vector for thedelivery of vaccine antigens and therapeutic agents to swine. Thefollowing properties of SPV support this rationale: SPV is only mildlypathogenic in swine, SPV is species specific, and SPV elicits aprotective immune response. Accordingly, SPV is an excellent candidatefor a viral vector delivery system, having little intrinsic risk whichmust be balanced against the benefit contributed by the vector's vaccineand therapeutic properties.

The prior art for this invention stems first from the ability to cloneand analyze DNA while in bacterial plasmids. The techniques that areavailable are detailed for the most part in Maniatis et al., 1983 andSambrook et al., 1989. These publications teach state of the art generalrecombinant DNA techniques.

Among the poxviruses, five (vaccinia, fowlpox, canarypox, pigeon, andraccoon pox) have been engineered, previous to this disclosure, tocontain foreign DNA sequences. Vaccinia virus has been used extensivelyto vector foreign genes (25) and is the subject of U.S. Pat. Nos.4,603,112 and 4,722,848. Similarly, fowlpox has been used to vectorforeign genes and is the subject of several patent applications EPA 0284 416, PCT WO 89/03429, and PCT WO 89/12684. Raccoon pox (10) andCanarypox (31) have been utilized to express antigens from the rabiesvirus. These examples of insertions of foreign genes into poxviruses donot include an example from the genus Suipoxvirus. Thus, they do notteach methods to genetically engineer swinepox viruses, that is, whereto make insertions and how to get expression in swinepox virus.

The idea of using live viruses as delivery systems for antigens has avery long history going back to the first live virus vaccines. Theantigens delivered were not foreign but were naturally expressed by thelive virus in the vaccines. The use of viruses to deliver foreignantigens in the modern sense became obvious with the recombinantvaccinia virus studies. The vaccinia virus was the vector and variousantigens from other disease causing viruses were the foreign antigens,and the vaccine was created by genetic engineering. While the conceptbecame obvious with these disclosures, what was not obvious was theanswer to a more practical question of what makes the best candidatevirus vector. In answering this question, details of the pathogenicityof the virus, its site of replication, the kind of immune response itelicits, the potential it has to express foreign antigens, itssuitability for genetic engineering, its probability of being licensedby regulatory agencies, etc, are all factors in the selection. The priorart does not teach these questions of utility.

The prior art relating to the use of poxviruses to deliver therapeuticagents relates to the use of a vaccinia virus to deliver interleukin-2(12). In this case, although the interleukin-2 had an attenuating effecton the vaccinia vector, the host did not demonstrate any therapeuticbenefit.

The therapeutic agent that is delivered by a viral vector of the presentinvention must be a biological molecule that is a by-product of swinepoxvirus replication. This limits the therapeutic agent in the firstanalysis to either DNA, RNA or protein. There are examples oftherapeutic agents from each of these classes of compounds in the formof anti-sense DNA, anti-sense RNA (16), ribozymes (34), suppressor tRNAs(2), interferon-inducing double stranded RNA and numerous examples ofprotein therapeutics, from hormones, e.g., insulin, to lymphokines,e.g., interferons and interleukins, to natural opiates. The discovery ofthese therapeutic agents and the elucidation of their structure andfunction does not make obvious the ability to use them in a viral vectordelivery system.

SUMMARY OF THE INVENTION

This invention provides a recombinant swinepox virus comprising aforeign DNA sequence inserted into the swinepox virus genomic DNA,wherein the foreign DNA sequence is inserted within a HindIII M fragmentof the swinepox virus genomic DNA and is capable of being expressed in aswinepox virus infected host cell.

This invention further provides homology vectors, vaccines and methodsof immunization.

BRIEF DESCRIPTION OF THE INVENTION

FIGS. 1A-1B: Show a detailed diagram of SPV genomic DNA (Kasza strain)including the unique long and Terminal repeat (TR) regions. Arestriction map for the enzyme HindIII is indicated (23). Fragments arelettered in order of decreasing size. Note that the terminal repeats aregreater than 2.1 kb but less than 9.7 kb in size.

FIGS. 2A-2B: Show the DNA sequence from homology vector 515-85.1. Thesequence of two regions of the homology vector 515-85.1 are shown. Thefirst region (FIG. 2A) (SEQ ID NO:1) covers a 599 base pair sequencewhich flanks the unique AccI site as indicated in FIGS. 3A-3C. Thebeginning (Met) and end (Val) of a 115 amino acid ORF is indicated bythe translation of amino acids below the DNA sequence. The second region(FIG. 2E) (SEQ ID NO:3) covers the 899 base pairs upstream of the uniqueHindIII site as indicated in FIGS. 3A-3C. The beginning (Asp) and end(Ile) of a 220 amino acid ORF is indicated by the translation of aminoacids below the DNA sequence.

FIGS. 3A-3C: Show the homology which exists between the 515.85.1 ORF andthe Vaccinia virus 01L ORF. FIG. 3A shows two maps: The first line ofFIG. 3A is a restriction map of the SPV HindIII M fragment and thesecond is a restriction map of the DNA insertion in plasmid 515-85.1.The location of the 515-85.1 [VV 01L-like] ORF is also indicated on themap. The locations of the DNA sequences shown in FIGS. 3B and 3C areindicated below the map by heavy bars in FIG. 3A. FIG. 3B shows thehomology between the VV 01L ORF (SEQ ID NO:5) and the 515-85.1 ORF (SEQID NO:6) at their respective N-termini. FIG. 3C shows the homologybetween the VV 01L ORF (SEQ ID NO:7) and the 515-85.1 ORF (SEQ ID NO:8)at their respective C-termini.

FIGS. 4A-4C: Show a description of the DNA insertion in Homology Vector520-17.5. FIG. 4A contains a diagram showing the orientation of DNAfragments assembled in plasmid 520-17.5 and table indicating the originof each fragment. FIG. 4B shows the sequences located at each of thejunctions A and B between fragments, and FIG. 4C shows the sequenceslocated at Junctions C and D (SEQ ID NO's: 9, 10, 13, and 16). FIGS. 4Band 4C further describe the restriction sites used to generate eachfragment as well as the synthetic linker sequences which were used tojoin the fragments are described for each junction. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements are also given. The following twoconventions are used: numbers in parenthesis ( ) refer to amino acids,and restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used, swinepox virus (SPV), early promoter 1 (EP1), late promoter 2(LP2), lactose operon Z gene (lacZ), and Escherichia coli (E. coli).

FIGS. 5A-5D: Show a detailed description of the DNA insertion inHomology Vector 538-46.16. FIG. 5A contains a diagram showing theorientation of DNA fragments assembled in plasmid 538-46.16 and a tableindicating the origin of each fragment. FIG. 5B shows the sequenceslocated at Junctions A and B between fragments, FIG. 5C shows sequenceslocated at Junction C and FIG. 5D shows sequences located at Junctions Dand E (SEQ ID NO's: 17, 18, 21, 26, and 28). FIGS. 5B to 5D alsodescribe the restriction sites used to generate each fragment as well asthe synthetic linker sequences which were used to join the fragments aredescribed for each junction. The synthetic linker sequences areunderlined by a heavy bar. The location of several gene coding regionsand regulatory elements is also given. The following two conventions areused: numbers in parenthesis ( ) refer to amino acids, and restrictionsites in brackets [ ] indicate the remnants of sites which weredestroyed during construction. The following abbreviations are used,swinepox virus (SPV), pseudorabies virus (PRV), g50 (gD), glycoprotein63 (g63), early promoter 1 (EP1), late promoter 1 (LP1) (SEQ ID NO: 46),late promoter 2 (LP2), lactose operon Z gene (lacZ), and Escherichiacoli (E. coli).

FIG. 6: Western blot of lysates from recombinant SPV infected cells withanti-serum to PRV. Lanes (A) uninfected Vero cell lysate, (B) S-PRV-000(pseudorabies virus S62/26) infected cell lysate, (C) pre-stainedmolecular weight markers, (D) uninfected EMSK cell lysate, (E) S-SPV-000infected cell lysate, (F) S-SPV-003 infected cell lysate, (G) S-SPV-008infected cell lysate. Cell lysates were prepared as described in thePREPARATION OF INFECTED CELL LYSATES. Approximately ⅕ of the totallysate sample was loaded in each lane.

FIG. 7: DNA sequence of NDV Hemagglutinin-Neuraminidase gene (HN) (SEQID NO: 29). The sequence of 1907 base pairs of the NDV HN cDNA clone areshown. The translational start and stop of the HN gene is indicated bythe amino acid translation below the DNA sequence.

FIGS. 8A-8D: Show a detailed description of the DNA insertion inHomology Vector 538-46.26. FIG. 5A contains a diagram showing theorientation of DNA fragments assembled in plasmid 538-46.26 and tableindicating the origin of each fragment. FIG. 8B shows the sequenceslocated at Junctions A and P between fragments; FIG. 8C shows thesequences located at Junctions C and D, FIG. 8D shows the sequenceslocated at Junction E (SEQ ID NO's: 31, 32, 34, 37, and 40). Therestriction sites used to generate each fragment as well as thesynthetic linker sequences which were used to join the fragments aredescribed for each junction in FIGS. 8B and 8D. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements is also given. The following twoconventions are used: numbers in parenthesis ( ) refer to amino acids,and restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used, swinepox virus (SPV), Newcastle Disease virus (NDV),hemagglutinin-neuraminidase (HN), early promoter 1 (EP1), late promoter1 (LP1), late promoter 2 (LP2), lactose operon Z gene (lacZ), andEscherichia coli (E. coli).

FIGS. 9A-9C: Show a detailed description of Swinepox Virus S-SPV-010 andthe DNA insertion in Homology Vector 561-36.26. FIG. 9A contains adiagram showing the orientation of DNA fragments assembled in plasmid561-36.26 and a table indicating the origin of each fragment. FIG. 9Bshows the sequences located at Junctions A and B between fragments andFIG. 9C show the sequences located at junction C and D (SEQ ID. NO: 47,48, 49, 50). The restriction sites used to generate each fragment aswell as synthetic linker sequences which are used to join the fragmentsare described for each junction in FIGS. 9B and 9C. The location ofseveral gene coding regions and regulatory elements is also given. Thefollowing two conventions are used: numbers in parentheses, ( ), referto amino acids, acid restriction sites in brackets, [ ], indicate theremnants of sites which are destroyed during construction. The followingabbreviations are used: swinepox virus (SPV), Escherichia coli (E.coli), thymidine kinase (TK), pox synthetic late promoter 1 (LP1), basepairs (BP).

FIGS. 10A-10D: Show a detailed description of Swinepox Virus S-SPV-011and the DNA insertion in Homology Vector 570-91.21. FIG. 10A contains adiagram showing the orientation of DNA fragments assembled in plasmid570-91.21 and a table indicating the origin of each fragment. FIG. 10Bshow the sequences located at Junctions A and B between fragments; FIG.10C shows the sequences located at Junction C, and FIG. 10D shows thesequences located at Junctions 10D and 10E (SEQ ID NOs: 51, 52, 53, 54,55). The restriction sites used to generate each fragment. as well assynthetic linker sequences which are used to join the fragments aredescribed for each junction in FIGS. 10B to 10D. The location of severalgene coding regions and regulatory elements is also given. The followingtwo conventions are used: S numbers in parentheses, ( ), refer to aminoacids, and restriction sites in brackets, [ ], indicate the remnants ofsites which are destroyed during construction. The followingabbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV),Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), poxsynthetic early promoter 2 (EP2) (SEQ ID NO: 45), gIII (gC), base pairs(BP).

FIGS. 11A-11D: Show a detailed description of Swinepox Virus S-SPV-012and the DNA insertion in Homology Vector 570-91.41. FIG. 11A contains adiagram showing the orientation of DNA fragments assembled in plasmid570-91.41 and a table indicating the origin of each fragment. FIG. 11Bshows the sequences located at Junctions A and B between fragments, FIG.11C shows the sequences located at Junction C, and FIG. 11D shows thesequence located at Junctions D and E. (SEQ ID NOs: 56, 57, 58, 59, 60).The restriction sites used to generate each fragment as well assynthetic linker sequences which are used to join the fragments aredescribed for each junction in FIGS. 11B to 11D. The location of severalgene coding regions and regulatory elements is also given. The followingtwo conventions are used: numbers in parentheses, ( ), refer to aminoacids, and restriction sites brackets, [ ], indicate the remnants ofsites which are destroyed during construction. The followingabbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV),Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), poxsynthetic early promoter 1 late promoter 2 (EP1LP2) (SEQ ID NO: 43),gIII (gC), base pairs (BP).

FIGS. 12A-12D: Show a detailed description of Swinepox Virus S-PRV-013and the DNA insertion in Homology Vector 570-91.64. FIG. 12A contains adiagram showing the orientation of DNA fragments assembled in plasmid570-91.64 and a table indicating the origin of each fragment. FIG. 12Bshows the sequences located at Junctions A and B between fragments, FIG.12C shows the sequences located at Junction C, and FIG. 12D shows thesequences located at Junctions D and E (SEQ ID NOs: 61, 62, 63, 64, 65).The restriction sites used to generate each fragment as well assynthetic linker sequences which are used to join the fragments aredescribed for each junction in FIGS. 12B to 12D. The location of severalgene coding regions and regulatory elements is also given. The followingtwo conventions are used: numbers in parentheses, ( ), refer to aminoacids, and restriction sites in brackets, [ ], indicate the remnants ofsites which are destroyed during construction. The followingabbreviations are used: swinepox virus (SPV), pseudorabies virus (PRV),Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), poxsynthetic late promoter 2 early promoter 2 (LP2EP2) (SEQ ID NO: 44),gIII (gC) base pairs (BP).

FIGS. 13A-13D: Show a detailed description of Swinepox Virus S-PRV-014and the DNA insertion in Homology Vector 599-65.25. FIG. 13A contains adiagram showing the orientation of DNA fragments assembled in plasmid599-65.25 and a table indicating the origin of each fragment. FIG. 13Bshows sequences located at Junctions A and B between the fragments, FIG.13C shows sequences located at Junction C, and FIG. 13D shows sequenceslocated at Junctions D and E. (SEQ ID NOs: 66, 67, 68, 69, and 70). Therestriction sites used to generate each fragment as well as syntheticlinker sequences which are used to join the fragments are described foreach junction in FIGS. 13B to 13D. The location of several gene codingregions and regulatory elements is also given. The following twoconventions are used: numbers in parentheses, ( ), refer to amino acids,and restriction sites in brackets, [ ], indicate the remnants of siteswhich are destroyed during construction. The following abbreviations areused: swinepox virus (SPV), infectious laryngotracheitis virus (ILT),Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), poxsynthetic early promoter 1 late promoter 2 (EP1LP2), glycoprotein G(gG), polymerase chain reaction (PCR), base pairs (BP).

FIGS. 14A-14D: Show a detailed description of Swinepox Virus S-SPV-016and the DNA insertion in Homology Vector 624-20.1C. FIG. 14A contains adiagram showing the orientation of DNA fragments assembled in plasmid624-20.1C and a table indicating the origin of each fragment. FIG. 14Bshows the sequences located at Junctions A and B between fragments; FIG.14C shows the sequences located at Junction C, and FIG. 14D shows thesequences at Junctions D and E. (SEQ ID NOs: 71, 72, 73, 74, and 75).The restriction sites are used to generate each fragment as well assynthetic linker sequences which are used to join the fragments aredescribed for each junction in FIGS. 14B to 14D. The location of severalgene coding regions and regulatory elements is also given. The followingtwo conventions are used: numbers in parentheses, ( ), refer to aminoacids, and restriction sites in brackets, [ ], indicate the remnants ofsites which are destroyed during construction. The followingabbreviations are used: swinepox virus (SPV), infectiouslaryngotracheitis virus (ILT), Escherichia coli (E. coli), pox syntheticlate promoter 1 (LP1), pox synthetic late promoter 2 early promoter 2(LP2EP2), glycoprotein I (gI), polymerase chain reaction (PCR), basepairs (BP).

FIGS. 15A-15D: Show a detailed description of Swinepox Virus S-SPV-017and the DNA insertion in Homology Vector 614-83.18. FIG. 15A contains adiagram showing the orientation of DNA fragments assembled in plasmid614-83.18 and a table showing the origin of each fragment. FIG. 15Bshows the sequences located at Junctions A and B between fragments, FIG.15C shows the sequences at Junction C, and FIG. 15D shows the sequenceslocated at Junctions D and E. The restriction sites used to generateeach fragment as well as synthetic linker sequences which are used tojoin the fragments are described for each junction in FIGS. 15B to 15D.The location of several gene coding regions and regulatory elements isalso given. The following two conventions are used: numbers inparentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), infectious bovine rhinotracheitis virus (IBR), Escherichia coli(E. coli), pox synthetic late promoter 1 (LP1), pox synthetic latepromoter 2 early promoter 2 (LP2EP2), glycoprotein G (gG), polymerasechain reaction (PCR), base pairs (BP).

FIG. 16: Western blot of lysates from recombinant SPV infected cellswith polyclonal goat anti-PRV gIII (gC). Lanes (A) S-PRV-002 (U.S. Pat.No. 4,877,737, issued Oct. 31, 1989) infected cell lysate, (B) molecularweight markers, (C) mock-infected EMSK cell lysate, (D) S-SPV-003infected cell lysate, (E) S-SPV-008 infected cell lysate, (F) S-SPV-011infected cell lysate, (G) S-SPV-012 infected cell lysate, (H) S-SPV-013infected cell lysate. Cell lysates are prepared as described in thePREPARATION OF INFECTED CELL LYSATES. Approximately ⅕ of the totallysates sample is loaded in each lane.

FIG. 17: Map showing the 5.6 kilobase pair HindIII M swinepox virusgenomic DNA fragment. Open reading frames (ORF) are shown with thenumber of amino acids coding in each open reading frame. The swinepoxvirus ORFs show significant sequence identities to the vaccinia virusORFs and are labeled with the vaccinia virus nomenclature (56 and 58).The I4L ORF (SEQ ID NO: 196) shows amino acid sequence homology toribonucleotide reductase large subunit (57), and the 01L ORF (SEQ ID NO:193) shows amino acid sequence homology to a leucine zipper motifcharacteristic of certain eukaryotic transcriptional regulatory proteins(13). The BglII site in the I4L ORF and the AccI site in the 01L ORF areinsertion sites for foreign DNA into non-essential regions of theswinepox genome. The homology vector 738-94.4 contains a deletion of SPVDNA from nucleotides 1679 to 2452 (SEQ ID NO: 189). The black bar at thebottom indicates regions for which the DNA sequence is known andreferences the SEQ ID NOs: 189 and 195. Positions of restriction sitesAccI, BglII, and HindIII are shown. I3L ORF (SEQ ID NO: 190), I2L ORF(SEQ ID NO: 191) and E1OR ORF (SEQ ID NO: 194) are shown.

FIGS. 18A-18D: Show a detailed description of Swinepox Virus S-SPV-034and the DNA insertion in Homology Vector 723-59A9.22. FIG. 18A containsa diagram showing the orientation of DNA fragments assembled in plasmid723-59A9.22 and a table indicating the origin of each fragment. FIG. 18Bshows the sequences located at Junctions A and B between fragments, FIG.18C shows the sequences located at Junction C, and FIG. 18D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 18Bto 18D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), equine influenza virus (EIV), Escherichia coli (E. coli), poxsynthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), neuraminidase (NA), Prague (PR), polymerase chainreaction (PCR), base pairs (BP).

FIGS. 19A-19D: Show a detailed description of Swinepox Virus S-SPV-015and the DNA insertion in Homology Vector 727-54.60. FIG. 19A contains adiagram showing the orientation of DNA fragments assembled in plasmid727-54.60 and a table indicating the origin of each fragment. FIG. 19Bshows the sequences located at Junctions A and B between fragments, FIG.19C shows the sequences located at Junction C, and FIG. 19D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 19Bto 19D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), pseudorabies virus (PRV), Escherichia coli (E. coli), poxsynthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), glycoprotein B (gB), base pairs (BP).

FIGS. 20A-20D: Show a detailed description of Swinepox Virus S-SPV-031and the DNA insertion in Homology Vector 7:27-67.18. FIG. 20A contains adiagram showing the orientation of DNA fragments assembled in plasmid727-67.18 and a table indicating the origin of each fragment. FIG. 20Bshows the sequences located at Junctions A and B between fragments, FIG.20C shows the sequences located at Junction C, and FIG. 20D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 20Bto 20D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1),pox synthetic early promoter 1 late promoter 2 (EP1LP2), antigen (Ag),base pairs (BP).

FIGS. 21A-21D: Show a detailed description of Swinepox Virus S-SPV-033and the DNA insertion in Homology Vector 732-18.4. FIG. 21A contains adiagram showing the orientation of DNA fragments assembled in plasmid732-18.4 and a table indicating the origin of each fragment. FIG. 21Bshows the sequences located at Junctions A and B between fragments, FIG.21C shows the sequences located at Junction C, and FIG. 21D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 21Bto 21D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1),pox synthetic late promoter 2 early promoter 2 (LP2EP2), equineinfluenza virus (EIV), neuraminidase (NA), Alaska (AK), polymerase chainreaction (PCR), base pairs (BP).

FIGS. 22A-22C: Show a detailed description of Swinepox Virus S-SPV-036and the DNA insertion in Homology Vector 741-80.3. FIG. 22A contains adiagram showing the orientation of DNA fragments assembled in plasmid741-80.3 and a table indicating the origin of each fragment. FIG. 22Bshows the sequences located at Junctions A, B, and C between fragmentsand FIG. 22C shows the sequences located at Junctions D, E and F. Therestriction sites used to generate each fragment as well as syntheticlinker sequences which are used to join the fragments are described foreach junction in FIGS. 22B and 22C. The location of several gene codingregions and regulatory elements is also given. The following twoconventions are used: numbers in parentheses, ( ), refer to amino acids,and restriction sites in brackets, [ ], indicate the remnants of siteswhich are destroyed during construction. The following abbreviations areused: swinepox virus (SPV), pseudorabies virus (PRV), Escherichia coli(E. coli), human cytomegalovirus immediate early (HCMV IE), poxsynthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), polyadenylation site (poly A), base pairs (BP).

FIGS. 23A-23D: Show a detailed description of Swinepox Virus S-SPV-035and the DNA insertion in Homology Vector 741-84.14. FIG. 23A contains adiagram showing the orientation of DNA fragments assembled in plasmid741-84.14 and a table indicating the origin of each fragment. FIG. 23Bshows the sequences located at Junctions A and B between fragments, FIG.23C shows the sequences located at Junction C, and FIG. 23D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 23Bto 23D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), pseudorabies virus (PRV), Escherichia coli (E. coli), poxsynthetic late promoter 1 (LP1) , pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), interleukin-2 (IL-2), glycoprotein X (gX)polymerase chain reaction (PCR), sequence (seq), base pairs (BP).

FIGS. 24A-24D: Show a detailed description of Swinepox Virus S-SPV-038and the DNA insertion in Homology Vector 744-34. FIG. 24A contains adiagram showing the orientation of DNA fragments assembled in plasmid744-34 and a table indicating the origin of each fragment. FIG. 24Bshows the sequences located at Junction A and B between fragments, FIG.24C shows the sequences located at Junction C, and FIG. 24D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 24Band 24D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), equine herpesvirus type 1 (EHV-1), Escherichia coli (E. coli),pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), glycoprotein B (gB), polymerase chain reaction(PCR), base pairs (BP).

FIGS. 25A-25D: Show a detailed description of Swinepox Virus S-SPV-039and the DNA insertion in Homology Vector 744-38. FIG. 25A contains adiagram showing the orientation of DNA fragments assembled in plasmid744-38 and a table indicating the origin of each fragment. FIG. 25Bshows the sequences located at Junction A and B between fragments. FIG.25C shows the sequences located at Junction C and FIG. 25D shows thesequences located at Junctions D and E. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction in FIGS. 25Bto 25D. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), equine herpesvirus type 1 (EHV-1), Escherichia coli (E. coli),pox synthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), glycoprotein D (gD), polymerase chain reaction(PCR), base pairs (BP).

FIGS. 26A-26D: Detailed description of Swinepox Virus S-SPV-042 and theDNA insertion in Homology Vector 751-07.A1. Diagram showing theorientation of DNA fragments assembled in plasmid 751-07.A1. The originof each fragment is indicated in the table. The sequence located at eachof the junctions between fragments is also shown. The restriction sitesused to generate each fragment as well as synthetic linker sequenceswhich are used to join the fragments are described for each junction.FIGS. 26A-26D show the sequences located at Junction A (SEQ ID NOS:197), (SEQ ID NO: 198), C (SEQ ID NO: 199), D (SEQ ID NO: 200) and E(SEQ ID NO: 201) between fragments and the sequences located at thejunctions. The location of several gene coding regions and regulatoryelements is also given. The following two conventions are used: numbersin parentheses, ( ), refer to amino acids, and restriction sites inbrackets, [ ], indicate the remnants of sites which are destroyed duringconstruction. The following abbreviations are used: swinepox virus(SPV), chicken interferon (cIFN), Escherichia coli (E. coli), poxsynthetic late promoter 1 (LP1), pox synthetic late promoter 2 earlypromoter 2 (LP2EP2), polymerase chain reaction (PCR), base pairs (BP).

FIGS. 27A-27D: Detailed description of Swinepox Virus S-SPV-043 and theDNA insertion in Homology Vector 751-56.A1. Diagram showing theorientation of DNA fragments assembled in plasmid 751-56.A1. The originof each fragment is indicated in the table. The sequences located ateach of the junctions between fragments is also shown. FIGS. 27A-27Dshow the sequences located at Junction A (SEQ ID NOS: 202), (SEQ ID NO:203), C (SEQ ID NO: 204), D (SEQ ID NO: 205) and E (SEQ ID NO: 206)between fragments and the sequences located at the junctions. Therestriction sites used to generate each fragment as well as syntheticlinker sequences which are used to join the several gene coding regionsand regulatory elements is also given. The following two conventions areused: numbers in parentheses, ( ), refer to amino acids, and restrictionsites in brackets, [ ], indicate the remnants of sites which aredestroyed during construction. The following abbreviations are used:swinepox virus (SPV), chicken myelomonocytic growth factor (cMGF),Escherichia coli (E. coli), pox synthetic late promoter 1 (LP1), poxsynthetic late promoter 2 early promoter 2 (LPE2EP2), polymerase chainreaction (PCR), base pairs (BP).

FIGS. 28A-28D: Detailed description of Swinepox Virus S-SPV-043 and theDNA insertion in Homology Vector 752-22.1. Diagram showing theorientation of DNA fragments assembled in plasmid 752-22.1. The originof each fragment is indicated in the table. The sequences located ateach of the junctions between fragments is also shown. FIGS. 28A-28Dshow the sequences located at Junction A (SEQ ID NOS: 207), (SEQ ID NO:208), C (SEQ ID NO: 209), and D (SEQ ID NO: 210) between fragments andthe sequences located at the junctions. The restriction sites used togenerate each fragment as well as synthetic linker sequences which areused to join the fragments are described for each junction. The locationof several gene coding regions and regulatory elements is also given.The following two conventions are used: numbers in parentheses, ( ),refer to amino acids, and restrictions sites in brackets, [ ], indicatethe remnants of sites which are destroyed during construction. Thefollowing abbreviations are used: swinepox virus (SPV), Escherichia coli(E. coli), pox synthetic late promoter 2 early promoter 2 (LP2EP2),polymerase chain reaction (PCR), bases pairs (BP).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant swinepox virus comprising aforeign DNA sequence inserted into the swinepox virus genomic DNA,wherein the foreign DNA sequence is inserted within a HindIII M fragmentof the swinepox virus genomic DNA and is capable of being expressed in aswinepox virus infected host cell.

In one embodiment the foreign DNA sequence of the recombinant swinepoxvirus is inserted within a non-essential Open Reading Frame (ORF) of theHindIII M fragment. Example of ORF's include, but are not limited to:I4L, I2L, 01L, and E10L.

In another embodiment the foreign DNA sequence of the recombinantswinepox virus is inserted within an approximately 2 Kb HindIII to BglIIsubfragment of the HindIII M fragment of the swinepox virus genomic DNA.In a preferred embodiment the foreign DNA sequence is inserted within aBglII site located within the approximately 2 Kb HindIII to BglIIsubfragment of the swinepox virus genomic DNA.

In another embodiment the foreign DNA sequence is inserted within alarger HindIII to BglII subfragment of the HindIII M fragment of theswinepox virus genomic DNA. In a preferred embodiment the foreign DNAsequence is inserted within an AccI site located within the largerHindIII to BglII subfragment of the swinepox virus genomic DNA.

In another embodiment the recombinant swinepox virus further comprises aforeign DNA sequence inserted into an open reading frame encodingswinepox virus thymidine kinase. In one embodiment the foreign DNAsequence is inserted into a NdeI site located within the open readingframe encoding the swinepox virus thymidine kinase.

For purposes of this invention, “a recombinant swinepox virus capable ofreplication” is a live swinepox virus which has been generated by therecombinant methods well known to those of skill in the art, e.g., themethods set forth in HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV in Materials and Methods and has not had geneticmaterial essential for the replication of the recombinant swinepox virusdeleted.

For purposes of this invention, “an insertion site which is notessential for replication of the swinepox virus” is a location in theswinepox viral genome where a sequence of DNA is not necessary for viralreplication, for example, complex protein binding sequences, sequenceswhich code for reverse transcriptase or an essential glycoprotein, DNAsequences necessary for packaging, etc.

For purposes of this invention, a “promoter” is a specific DNA sequenceon the DNA molecule to which the foreign RNA polymerase attaches and atwhich transcription of the foreign RNA is initiated.

For purposes of this invention, an “open reading frame” is a segment ofDNA which contains codons that can be transcribed into RNA which can betranslated into an amino acid sequence and which does not contain atermination codon.

In addition, the present invention provides a recombinant swinepox virus(SPV) capable of replication in an animal into which the recombinantswinepox virus is introduced which comprises swinepox viral DNA andforeign DNA encoding RNA which does not naturally occur in the animalinto which the recombinant swinepox virus is introduced, the foreign DNAbeing inserted into the swinepox viral DNA at an insertion site which isnot essential for replication of the swinepox virus and being under thecontrol of a promoter.

The invention further provides a foreign DNA sequence or foreign RNAwhich encodes a polypeptide. Preferably, the polypeptide is antigenic inthe animal. Preferably, this antigenic polypeptide is a linear polymerof more than 10 amino acids linked by peptide bonds which stimulates theanimal to produce antibodies.

The invention further provides a recombinant swinepox virus capable ofreplication which contains a foreign DNA encoding a polypeptide which isa detectable marker. Preferably the detectable marker is the polypeptideE. coli β-galactosidase. Preferably, the insertion site for the foreignDNA encoding E. coli β-galactosidase is the AccI restrictionendonuclease site located within the HindIII M fragment of the swinepoxviral DNA. Preferably, this recombinant swinepox virus is designatedS-SPV-003 (ATCC Accession No. VR 2335). The S-SPV-003 swinepox virus hasbeen deposited pursuant to the Budapest Treaty on the InternationalDeposit of Microorganisms for the Purposes of Patent Procedure with thePatent Culture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR2335.

For purposes of this invention, a “polypeptide which is a detectablemarker” includes the bimer, trimer and tetramer form of the polypeptide.E. coli β-galactosidase is a tetramer composed of four polypeptides ormonomer sub-units.

The invention further provides a recombinant swinepox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is or is from pseudorabies virus (PRV) g950 (gD), pseudorabiesvirus (PRV) gII (gB), Pseudorabies virus (PRV) gIII (gC), pseudorabiesvirus (PRV) glycoprotein H, pseudorabies virus (PRV) glycoprotein E,Transmissible gastroenteritis (TGE) glycoprotein 195, Transmissiblegastroenteritis (TGE) matrix protein, swine rotavirus glycoprotein 38,swine parvovirus capsid protein, Serpulina hydodysenteriae protectiveantigen, Bovine Viral Diarrhea (BVD) glycoprotein 55, Newcastle DiseaseVirus (NDV) hemagglutinin-neuraminidase, swine flu hemagglutinin orswine flu neuraminidase. Preferably, the antigenic polypeptide isPseudorabies Virus (PRV) g50 (gD). Preferably, the antigenic protein isNewcastle Disease Virus (NDV) hemagglutinin-neuraminidase.

The invention further provides a recombinant swinepox virus capable ofreplication which contains foreign DNA encoding an antigenic polypeptidewhich is or is from Serpulina hyodysenteriae, Foot and Mouth DiseaseVirus, Hog Cholera Virus, Swine Influenza Virus, African Swine FeverVirus or Mycoplasma hyopneumoniae.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) g50 (gD). This recombinant swinepox virus can be furtherengineered to contain foreign DNA encoding a detectable marker, such asE. coli β-galactosidase. A preferred site within the swinepox viralgenome for insertion of the foreign DNA encoding PRV g50 (gD) and E.coli β-galactosidase is the AccI site within the HindIII M fragment ofthe swinepox viral DNA. Preferably, this recombinant swinepox virus isdesignated S-SPV-008 (ATCC Accession No. VR 2339). The S-SPV-008swinepox virus has been deposited pursuant to the Budapest Treaty on theInternational Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A.under ATCC Accession No. VR 2339.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) gIII (gC). This recombinant swinepox virus can also be furtherengineered to contain foreign DNA encoding a detectable marker, such asE. coli β-galactosidase. A preferred site within the swinepox viral DNAfor insertion of the foreign DNA encoding PRV C gene and E. coliβ-galactosidase is the AccI site within the HindIII M fragment of theswinepox viral DNA. Preferably, this recombinant swinepox virus isdesignated S-SPV-011, S-SPV-012, or S-SPV-013. The swinepox virusdesignated S-SPV-013 has been deposited on Jul. 16, 1993 pursuant to theBudapest Treaty on the International Deposit of Microorganisms for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under ATCC Accession No. VR 2418.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) gII (gB). This recombinant swinepox virus can also be furtherengineered to contain foreign DNA encoding a detectable marker, such asE. coli β-galactosidase. A preferred site within the swinepox viral DNAfor insertion of the foreign DNA encoding PRV gII (gB) and E. coliβ-galactosidase is the AccI site within the HindIII M fragment of theswinepox viral DNA. Preferably, this recombinant swinepox virus isdesignated S-SPV-015 (ATCC Accession No. VR 2466). The S-SPV-015swinepox virus has been deposited on Jul. 22, 1994 pursuant to theBudapest Treaty on the International Deposit of Microorganisms for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under ATCC Accession No, VR 2466.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) g50 (gD) and foreign DNA encoding pseudorabies virus (PRV) gIII(gC). This recombinant swinepox virus can also be further engineered tocontain foreign DNA encoding a detectable marker, such as E. coliβ-galactosidase. A preferred site within the swinepox viral DNA forinsertion of the foreign DNA encoding PRV g50 (gD), PRV gIII (gC) and E.coli β-galactosidase is the AccI site within the HindIII M fragment ofthe swinepox viral DNA.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) g50 (gD) and foreign DNA encoding pseudorabies virus (PRV) gII(gB). This recombinant swinepox virus can also be further engineered tocontain foreign DNA encoding a detectable marker, such as E. coliβ-galactosidase. A preferred site within the swinepox viral genome forinsertion of foreign DNA encoding PRV g50 (gD), PRV gII (gB) and E. coliβ-galactosidase is the AccI site within the HindIII M fragment of theswinepox viral DNA.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) gIII (gC) and foreign DNA encoding pseudorabies virus (PRV) gII(gB). This recombinant swinepox virus can also be further engineered tocontain foreign DNA encoding a detectable marker, such as E. coliβ-galactosidase. A preferred site within the swinepox viral genome forinsertion of foreign DNA encoding PRV gIII (gC), PRV gII (gB) and E.coli β-galactosidase is the AccI site within the HindIII M fragment ofthe swinepox viral DNA.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding pseudorabies virus(PRV) g50 (gD), foreign DNA encoding pseudorabies virus (PRV) gIII (gC),and foreign DNA encoding pseudorabies virus (PRV) gII (gB). Thisrecombinant swinepox virus can also be further engineered to containforeign DNA encoding a detectable marker, such as E. coliβ-galactosidase.

A preferred site within the swinepox viral genome for insertion offoreign DNA encoding PRV g50 (gD), PRV gIII (gC), PRV gII (gB) and E.coli β-galactosidase is the AccI site within the HindIII M fragment ofthe swinepox viral DNA.

The invention further provides for a recombinant swinepox virus capableof replication which contains foreign DNA encoding RNA encoding theantigenic polypeptide Newcastle Disease Virus (NDV)hemagglutinin-neuraminidase further comprising foreign DNA encoding apolypeptide which is a detectable marker. Preferably, this recombinantswinepox virus is designated S-SPV-009 (ATCC Accession No. VR 2344). TheS-SPV-009 swinepox virus has been deposited pursuant to the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure with the Patent Culture Depository of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852U.S.A. under ATCC Accession No. VR 2344.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from infectious bovine rhinotracheitisvirus and is capable of being expressed in a host infected by therecombinant swinepox virus. Examples of such antigenic polypeptide areinfectious bovine rhinotracheitis virus glycoprotein E and glycoproteinG. Preferred embodiment of this invention are recombinant swinepoxviruses designated S-SPV-017 and S-SPV-019.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from infectious laryngotracheitis virusand is capable of being expressed in a host infected by the recombinantswinepox virus. Examples of such antigenic polypeptide are infectiouslaryngotracheitis virus glycoprotein G and glycoprotein I. Preferredembodiment of this invention are recombinant swinepox viruses designatedS-SPV-014 and S-SPV-016.

In one embodiment of the recombinant swinepox virus the foreign DNAsequence encodes a cytokine. In another embodiment the cytokine ischicken myelomonocytic growth factor (cMGF) or chicken interferon(cIFN). Cytokines include, but are not limited to: transforming growthfactor beta, epidermal growth factor family, fibroblast growth factors,hepatocyte growth factor, insulin-like growth factor, vascularendothelial growth factor, interleukin 1, IL-1 receptor antagonist,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, IL-6 soluble receptor, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12,interleukin-13, angiogenin, chemokines, colony stimulating factors,granulocyte-macrophage colony stimulating factors, erythropoietin,interferon, interferon gamma, c-kit ligand, leukemia inhibitory factor,oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stemcell factor, tumor necrosis factors, and soluble TNF receptors. Thesecytokines are from humans, bovine, equine, feline, canine, porcine oravian. Preferred embodiments of such recombinant virus are designatedS-SPV-042, and S-SPV-043.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from a human pathogen and is capable ofbeing expressed in a host infected by the recombinant swinepox virus.

Recombinant SPV expressing cytokines is used to enhance the immuneresponse either alone or when combined with vaccines containingcytokines or antigen genes of disease causing microorganisms.

Antigenic polypeptide of a human pathogen which are derived from humanherpesvirus include, but are not limited to: hepatitis B virus andhepatitis C virus hepatitis B virus surface and core antigens, hepatitisC virus, human immunodeficiency virus, herpes simplex virus-1, herpessimplex virus-2, human cytomegalovirus, Epstein-Barr virus,Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, humaninfluenza, measles virus, hantaan virus, pneumonia virus, rhinovirus,poliovirus, human respiratory syncytial virus, retrovirus, human T-cellleukemia virus, rabies virus, mumps virus, malaria (Plasmodiumfalciparum), Bordetella pertussis, Diptheria, Rickettsia prowazekii,Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.

In one embodiment of the invention, a recombinant swinepox viruscontains the foreign DNA sequence encoding hepatitis B virus coreprotein. Preferably, such virus recombinant virus is designatedS-SPV-031.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes acytokine capable of stimulating an immune in a host infected by therecombinant swinepox virus and is capable of being expressed in the hostinfected.

In one embodiment of the invention, a recombinant swinepox viruscontains a foreign DNA sequence encoding human interleukin-2.Preferably, such recombinant virus is designated S-SPV-035.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from an equine pathogen and is capable ofbeing expressed in a host infected by the recombinant swinepox virus.

The antigenic polypeptide of an equine pathogen can derived from equineinfluenza virus, or equine herpesvirus. In one embodiment the antigenicpolypeptide is equine influenza neuraminidase or hemagglutinin. Examplesof such antigenic polypeptide are equine influenza virus type A/Alaska91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase,equine influenza virus type A/Miami 63 neuraminidase, equine influenzavirus type A/Kentucky 81 neuraminidase, equine influenza virus typeA/Kentucky 92 neuraminidase equine herpesvirus type 1 glycoprotein B,equine herpesvirus type 1 glycoprotein D, Streptococcus equi, equineinfectious anemia virus, equine encephalitis virus, equine rhinovirusand equine rotavirus. Preferred embodiments of such recombinant virusare designated S-SPV-033, S-SPV-034, S-SPV-038, S-SPV-039 and S-SPV-041.

The present invention further provides an antigenic polypeptide whichincludes, but is not limited to: hog cholera virus gE1, hog choleravirus gE2, Swine influenza virus hemagglutinin, neurominidase, matrixand nucleoprotein, pseudorabies virus gB, gC and gD, and PRRS virusORF7.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from bovine respiratory syncytial virus orbovine parainfluenza virus, and is capable of being expressed in a hostinfected by the recombinant swinepox virus.

For example, the antigenic polypeptide of derived from infectious bovinerhinotracheitis virus gE, bovine respiratory syncytial virus equinepathogen can derived from equine influenza virus is bovine respiratorysyncytial virus attachment protein (BRSV G), bovine respiratorysyncytial virus fusion protein (BRSV F), bovine respiratory syncytialvirus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3fusion protein, and the bovine parainfluenza virus type 3 hemagglutininneuraminidase. In a preferred embodiment the recombinant swinepox virusis designated S-SPV-045.

Preferred embodiments of a recombinant virus containing a foreign DNAencoding an antigenic polypeptide from a bovine respiratory syncytialvirus are designated S-SPV-020, S-SPV-029, and S-SPV-030. And apreferred embodiment of a recombinant virus containing a foreign DNAencoding an antigenic polypeptide from a bovine parainfluenza virus aredesignated S-SPV-028.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodesbovine viral diarrhea virus (BVDV) glycoprotein 48 or glycoprotein 53,and wherein the foreign DNA sequence is capable of being expressed in ahost infected by the recombinant swinepox virus. Preferred embodimentsof such virus are designated S-SPV-032, S-SPV-040, S-SPV-049, andS-SPV-050.

The present invention further provides a recombinant swinepox viruswhich comprises a foreign DNA sequence inserted into a non-essentialsite of the swinepox genome, wherein the foreign DNA sequence encodes anantigenic polypeptide derived from infectious bursal disease virus andwherein the foreign DNA sequence is capable of being expressed in a hostinfected by the recombinant swinepox virus. Examples of such antigenicpolypeptide are infectious bursal disease virus polyprotein and VP2.Preferred embodiments of such virus are designated S-SPV-026 andS-SPV-027.

The present invention further provides a recombinant swinepox virus inwhich the foreign DNA sequence encodes an antigenic polypeptide whichincludes, but is not limited to: MDV gA, MDV gB, MDV gD, NDV HN, NDV F,ILT gB, ILT gI, ILT gD, IBDV VP2, IBDV VP3, IBDV VP4, IBDV polyprotein,IBV spike, IBV matrix, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia virus,Salmonella spp. E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., Poultry nematodes, cestodes,trematodes, poultry mites/lice, and poultry protozoa.

The invention further provides that the inserted foreign DNA sequence isunder the control of a promoter. In one embodiment the is a swinepoxviral promoter. In another embodiment the foreign DNA sequence is undercontrol of an endogenous upstream poxvirus promoter. In anotherembodiment the foreign DNA sequence is under control of a heterologousupstream promoter.

For purposes of this invention, promoters include but is not limited to:synthetic pox viral promoter, pox synthetic late promoter 1, poxsynthetic late promoter 2 early promoter 2, pox O1L promoter, pox I4Lpromoter, pox I3L promoter, pox I2L promoter, pox I1L promoter, pox E10Rpromoter, PRV gX, HSV-1 alpha 4, HCMV immediate early, MDV gA, MDV gB,MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD. Alternate promoters aregenerated by methods well known to those of skill in the art, forexample, as set forth in the STRATEGY FOR THE CONSTRUCTION OF SYNTHETICPOX VIRAL PROMOTERS in Materials and Methods.

The invention provides for a homology vector for producing a recombinantswinepox virus by inserting foreign DNA into the genomic DNA of aswinepox virus. The homology vector comprises a double-stranded DNAmolecule consisting essentially of a double-stranded foreign DNAsequence or (RNA) which does not naturally occur in an animal into whichthe recombinant swinepox virus is introduced, with at one end of theforeign DNA, double-stranded swinepox viral DNA homologous to genomicDNA located at one side of a site on the genomic DNA which is notessential for replication of the swinepox virus, and at the other end ofthe foreign DNA, double-stranded swinepox viral DNA homologous togenomic DNA located at the other side of the same site on the genomicDNA. Preferably, the RNA encodes a polypeptide.

In another embodiment of the present invention, the double-strandedswinepox viral DNA of the homology vectors described above is homologousto genomic DNA present within the HindIII M fragment. In anotherembodiment the double-stranded swinepox viral DNA of the homologyvectors described above is homologous to genomic DNA present within anapproximately 2 Kb HindIII to BglII sub-fragment. In a preferredembodiment the double-stranded swinepox viral DNA is homologous togenomic DNA present within the BglII site located in this HindIII toBglII subfragment.

In another embodiment the double-stranded swinepox viral DNA ishomologous to genomic DNA present within the open reading framecontained in the larger HindIII to BglII subfragment. Preferably, thedouble-stranded swinepox viral DNA is homologous to genomic DNA presentwithin the AccI restriction endonuclease site located in the largerHindIII to BglII subfragment.

In a preferred embodiment the homology vectors are designated 752-29.33,751-07.A1, 751-56.A1, 751-22.1, 746-94.1, 767-67.3, 738-94.4, and771-55.11.

In one embodiment, the polypeptide is a detectable marker. Preferably,the polypeptide which is a detectable marker is E. coli β-galactosidase.

In one embodiment, the polypeptide is antigenic in the animal.Preferably, the antigenic polypeptide is or is from pseudorabies virus(PRV) g50 (gD), pseudorabies virus (PRV) gII (gB), Pseudorabies virus(PRV) gIII (gC), Pseudorabies virus (PRV) glycoprotein H, Transmissiblegastroenteritis (TGE) glycoprotein 195, Transmissible gastroenteritis(TGE) matrix protein, swine rotavirus glycoprotein 38, swine parvoviruscapsid protein, Serpulina hydodysenteriae protective antigen, BovineViral Diarrhea (BVD) glycoprotein 53 and g48, Newcastle Disease Virus(NDV) hemagglutinin-neuraminidase, swine flu hemagglutinin or swine fluneuraminidase. Preferably, the antigenic polypeptide is or is fromSerpulina hyodysenteriae, Foot and Mouth Disease Virus, Hog CholeraVirus gE1 and gE2, Swine Influenza Virus, African Swine Fever Virus orMycoplasma hyopneumoniae, swine influenza virus hemagglutinin,neuraminidase and matrix and nucleoprotein, PRRS virus ORF7, andhepatitis B virus core protein.

In an embodiment of the present invention, the double stranded foreignDNA sequence in the homology vector encodes an antigenic polypeptidederived from a human pathogen.

For example, the antigenic polypeptide of a human pathogen is derivedfrom human herpesvirus, herpes simplex virus-1, herpes simplex virus-2,human cytomegalovirus, Epstein-Barr virus, Varicell-Zoster virus, humanherpesvirus-6, human herpesvirus-7, human influenza, humanimmunodeficiency virus, rabies virus, measles virus, hepatitis B virusand hepatitis C virus. Furthermore, the antigenic polypeptide of a humanpathogen may be associated with malaria or malignant tumor from thegroup consisting of Plasmodium falciparum, Bordetella pertusis, andmalignant tumor.

In an embodiment of the present invention, the double stranded foreignDNA sequence in the homology vector encodes a cytokine capable ofstimulating human immune response. In one embodiment the cytokine is achicken myelomonocytic growth factor (cMGF) or chicken interferon(cIFN). For example, the cytokine can be, but not limited to,interleukin-2, interleukin-6, interleukin-12, interferons,granulocyte-macrophage colony stimulating factors, and interleukinreceptors.

In an embodiment of the present invention, the double stranded foreignDNA sequence in the homology vector encodes an antigenic polypeptidederived from an equine pathogen.

The antigenic polypeptide of an equine pathogen can derived from equineinfluenza virus or equine herpesvirus. Examples of such antigenicpolypeptide are equine influenza virus type A/Alaska 91 neuraminidase,equine influenza virus type A/Prague 56 neuraminidase, equine influenzavirus type A/Miami 63 neuraminidase, equine influenza virus typeA/Kentucky 81 neuraminidaseequine herpesvirus type 1 glycoprotein B, andequine herpesvirus type 1 glycoprotein D.

In an embodiment of the present invention, the double stranded foreignDNA sequence of the homology vector encoded an antigenic polypeptidederived from bovine respiratory syncytial virus or bovine parainfluenzavirus.

For example, the antigenic polypeptide is derived from infectious bovinerhinotracheitis gE, bovine respiratory syncytial virus attachmentprotein (BRSV G), bovine respiratory syncytial virus fusion protein(BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSVN), bovine parainfluenza virus type 3 fusion protein, and the bovineparainfluenza virus type 3 hemagglutinin neuraminidase.

In an embodiment of the present invention, the double stranded foreignDNA sequence of the homology vector encodes an antigenic polypeptidederived from infectious bursal disease virus. Examples of such antigenicpolypeptide are infectious bursal disease virus polyprotein andinfectious bursal disease virus VP2, VP3, or VP4.

For purposes of this invention, a “homology vector” is a plasmidconstructed to insert foreign DNA in a specific site on the genome of aswinepox virus.

In one embodiment of the invention, the double-stranded swinepox viralDNA of the homology vectors described above is homologous to genomic DNApresent within the open reading frame encoding swinepox thymidinekinase. Preferably, the double-stranded swinepox viral DNA is homologousto genomic DNA present within the NdeI restriction endonuclease sitelocated in the open reading frame encoding swinepox thymidine kinase.

The invention further provides a homology vectors described above, theforeign DNA sequence of which is under control of a promoter locatedupstream of the foreign DNA sequence. The promoter can be an endogenousswinepox viral promoter or an exogenous promoter. Promoters include, butare not limited to: synthetic pox viral promoter, pox synthetic latepromoter 1, pox synthetic late promoter 2 early promoter 2, pox O1Lpromoter, pox I4L promoter, pox I3L promoter, pox I2L promoter, pox I1Lpromoter, pox E10R promoter, PRV gX, HSV-1 alpha 4, HCMV immediateearly, MDV gA, MDV gB, MDV gD, ILT gB, BHV-1.1 VP8 and ILT gD.

The invention further provides a vaccine which comprises an effectiveimmunizing amount of a recombinant swinepox virus of the presentinvention and a suitable carrier.

Suitable carriers for the swinepox virus are well known in the art andinclude proteins, sugars, etc. One example of such a suitable carrier isa physiologically balanced culture medium containing one or morestabilizing agents such as stabilized, hydrolyzed proteins, lactose,etc.

For purposes of this invention, an “effective immunizing amount” of therecombinant swinepox virus of the present invention is within the rangeof 10³ to 10⁹ PFU/dose.

The present invention also provides a method of immunizing an animal,wherein the animal is a human, swine, bovine, equine, caprine or ovine.For purposes of this invention, this includes immunizing the animalagainst the virus or viruses which cause the disease or diseasespseudorabies, transmissible gastroenteritis, swine rotavirus, swineparvovirus, Serpulina hyodysenteriae, bovine viral diarrhea, Newcastledisease, swine influenza, PRRS, bovine respiratory synctial virus,bovine parainfluenza virus type 3, foot and mouth disease, hog cholera,African swine fever or Mycoplasma hyopneumoniae. For purposes of thisinvention, the method of immunizing also includes immunizing the animalagainst human pathogens, bovine pathogens, equine pathogens, avianpathogens described in the preceding part of this section.

The method comprises administering to the animal an effective immunizingdose of the vaccine of the present invention. The vaccine may beadministered by any of the methods well known to those skilled in theart, for example, by intramuscular, subcutaneous, intraperitoneal orintravenous injection. Alternatively, the vaccine may be administeredintranasally or orally.

The present invention also provides a method for testing a swine todetermine whether the swine has been vaccinated with the vaccine of thepresent invention, particularly the embodiment which contains therecombinant swinepox virus S-SPV-008 (ATCC Accession No. VR 2339), or isinfected with a naturally-occurring, wild-type pseudorabies virus. Thismethod comprises obtaining from the swine to be tested a sample of asuitable body fluid, detecting in the sample the presence of antibodiesto pseudorabies virus, the absence of such antibodies indicating thatthe swine has been neither vaccinated nor infected, and for the swine inwhich antibodies to pseudorabies virus are present, detecting in thesample the absence of antibodies to pseudorabies virus antigens whichare normally present in the body fluid of a swine infected by thenaturally-occurring pseudorabies virus but which are not present in avaccinated swine indicating that the swine was vaccinated and is notinfected.

The present invention provides a recombinant SPV which when insertedwith a foreign DNA sequence or gene may be employed as a diagnosticassay. In one embodiment FIV env and gag genes and D. immitis p39 and 22kd are employed in a diagnostic assay to detect feline immunodeficiencycaused by FIV and to detect heartworm caused by D. immits, respectively.

The present invention also provides a host cell infected with arecombinant swinepox virus capable of replication. In one embodiment,the host cell is a mammalian cell. Preferably, the mammalian cell is aVero cell. Preferably, the mammalian cell is an ESK-4 cell, PK-15 cellor EMSK cell.

For purposes of this invention a “host cell” is a cell used to propagatea vector and its insert. Infecting the cells was accomplished by methodswell known to those of skill in the art, for example, as set forth inINFECTION—TRANSFECTION PROCEDURE in Material and Methods.

Methods for constructing, selecting and purifying recombinant swinepoxviruses described above are detailed below in Materials and Methods.

EXPERIMENTAL DETAILS Materials and Methods

Preparation of Swinepox Virus Stock Samples

Swinepox virus (SPV) samples were prepared by infecting embryonic swinekidney (EMSK) cells, ESK-4 cells, PK-15 cells or Vero cells at amultiplicity of infection of 0.01 PFU/cell in a 1:1 mixture of Iscove'sModified Dulbecco's Medium (IMDM) and RPMI 1640 medium containing 2 mMglutamine, 100 units/ml penicillin, 100 units/ml streptomycin (thesecomponents were obtained from Sigma or equivalent supplier, andhereafter are referred to as EMSK negative medium). Prior to infection,the cell monolayers were washed once with EMSK negative medium to removetraces of fetal bovine serum. The SPV contained in the initial inoculum(0.5 ml for 10 cm plate; 10 ml for T175 cm flask) was then allowed toabsorb onto the cell monolayer for two hours, being redistributed everyhalf hour. After this period, the original inoculum was brought up tothe recommended volume with the addition of complete EMSK medium (EMSKnegative medium plus 5% fetal bovine serum). The plates were incubatedat 37° C. in 5% CO₂ until cytopathic effect was complete. The medium andcells were harvested and frozen in a 50 ml conical screw cap tube at−70° C. Upon thawing at 37° C., the virus stock was aliquoted into 1.0ml vials and refrozen at −70° C. The titers were usually about 10⁶PFU/ml.

PREPARATION OF SPV DNA

For swinepox virus DNA isolation, a confluent monolayer of EMSK cells ina T175 cm² flask was infected at a multiplicity of 0.1 and incubated 4-6days until the cells were showing 100% cytopathic effect. The infectedcells were then harvested by scraping the cells into the medium andcentrifuging at 3000 rpm for 5 minutes in a clinical centrifuge. Themedium was decanted, and the cell pellet was gently resuspended in 1.0ml Phosphate Buffer Saline (PBS: 1.5g Na₂HPO₄, 0.2 g KH₂PO₄, 0.8 g NaCLand 0.2 g KCl per liter H₂O) (per T175) and subjected to two successivefreeze-thaws (−70° C. to 37° C.). Upon the last thaw, the cells (on ice)were sonicated two times for 30 seconds each with 45 seconds coolingtime in between. Cellular debris was then removed by centrifuging(Sorvall RC-5B superspeed centrifuge) at 3000 rpm for 5 minutes in a HB4rotor at 4° C. SPV virions, present in the supernatant, were thenpelleted by centrifugation at 15,000 rpm for 20 minutes at 4° C. in aSS34 rotor (Sorvall) and resuspended in 10 mM Tris (pH 7.5). Thisfraction was then layered onto a 36% sucrose gradient (w/v in 10 mM trispH 7.5) and centrifuged (Beckman L8-70M Ultracentrifuge) at 18,000 rpmfor 60 minutes in a SW41 rotor (Beckman) at 4° C. The virion pellet wasresuspended in 1.0 ml of 10 mM tris pH 7.5 and sonicated on ice for 30seconds. This fraction was layered onto a 20% to 50% continuous sucrosegradient and centrifuged 16,000 rpm for 60 minutes in a SW41 rotor at 4°C. The SPV virion band located about three quarters down the gradientwas harvested, diluted with 20% sucrose and pelleted by centrifugationat 18,000 rpm for 60 minutes in a SW41 rotor at 4° C. The resultantpellet was then washed once with 10 mM Tris pH 7.5 to remove traces ofsucrose and finally resuspended in 10 mM Tris pH 7.5. SPV DNA was thenextracted from the purified virions by lysis (4 hours at 60° C.) inducedby the addition of EDTA, SDS, and proteinase K to final concentrationsof 20 mM, 0.5. and 0.5 mg/ml, respectively. After digestion, threephenol:chloroform (1:1) extractions were conducted and the sampleprecipitated by the addition of two volumes of absolute ethanol andincubation at −20° C. for 30 minutes. The sample was then centrifuged inan Eppendorf minifuge for 5 minutes at full speed. The supernatant wasdecanted, and the pellet air dried and rehydrated in 0.01 M Tris pH 7.5,1 mM EDTA at 4° C.

PREPARATION OF INFECTED CELL LYSATES

For cell lysate preparation, serum free medium was used. A confluentmonolayer of cells (EMSK, ESK-4, PK-15 or Vero for SPV or VERO for PRV)in a 25 cm² flask or a 60 mm petri dish was infected with 100 μl ofvirus sample. After cytopathic effect was complete, the medium and cellswere harvested and the cells were pelleted at 3000 rpm for 5 minutes ina clinical centrifuge. The cell pellet was resuspended in 250 μl ofdisruption buffer (2% sodium dodecyl sulfate, 2% β-mercapto-ethanol).The samples were sonicated for 30 seconds on ice and stored at −20° C.

WESTERN BLOTTING PROCEDURE

Samples of lysates and protein standards were run on a polyacrylamidegel according to the procedure of Laemnli (1970). After gelelectrophoresis the proteins were transferred and processed according toSambrook et al. (1982). The primary antibody was a swine anti-PRV serum(Shope strain; lot370, PDV8201, NVSL, Ames, Iowa) diluted 1:100 with 5%non-fat dry milk in Tris-sodium chloride, and sodium Azide (TSA: 6.61 gTris-HCl, 0.97 g Tris-base, 9.0 g NaCl and 2.0 g Sodium Azide per literH₂O). The secondary antibody was a goat anti-swine alkaline phosphataseconjugate diluted 1:1000 with TSA.

MOLECULAR BIOLOGICAL TECHNIQUES

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described by Maniatis et al. (1982) and Sambrook et al.(1989). Except as noted, these were used with minor variation.

DNA SEQUENCING

Sequencing was performed using the USB Sequenase Kit and ³⁵S-DATP (NEN).Reactions using both the dGTP mixes and the dITP mixes were performed toclarify areas of compression. Alternatively, compressed areas wereresolved on formamide gels. Templates were double-stranded plasmidsubclones or single stranded M13 subclones, and primers were either madeto the vector just outside the insert to be sequenced, or to previouslyobtained sequence. Sequence obtained was assembled and compared usingDnastar software. Manipulation and comparison of sequences obtained wasperformed with Superclone™ and Supersee™ programs from Coral Software.

CLONING WITH THE POLYMERASE CEAIN REACTION

The polymerase chain reaction (PCR) was used to introduce restrictionsites convenient for the manipulation of various DNAS. The proceduresused are described by Innis, et al. (1990). In general, amplifiedfragments were less than 500 base pairs in size and critical regions ofamplified fragments were confirmed by DNA sequencing. The primers usedin each case are detailed in the descriptions of the construction ofhomology vectors below.

HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV

This method relies upon the homologous recombination between theswinepox virus DNA and the plasmid homology vector DNA which occurs inthe tissue culture cells containing both swinepox virus DNA andtransfected plasmid homology vector. For homologous recombination tooccur, the monolayers of EMSK cells are infected with S-SPV-001 (KaszaSPV strain, 17) at a multiplicity of infection of 0.01 PFU/cell tointroduce replicating SPV (i.e. DNA synthesis) into the cells. Theplasmid homology vector DNA is then transfected into these cellsaccording to the INFECTION—TRANSFECTION PROCEDURE. The construction ofhomology vectors used in this procedure is described below

INFECTION—TRANSFECTION PROCEDURE

6 cm plates of EMSK cells (about 80% confluent) were infected withS-SPV-001 at a multiplicity of infection of 0.01 PFU/cell in EMSKnegative medium and incubated at 37° C. in a humidified 5% CO₂environment for 5 hours. The transfection procedure used is essentiallythat recommended for Lipofectin™ Reagent (BRL). Briefly, for each 6 cmplate, 15 μg of plasmid DNA was diluted up to 100 μl with H₂O.Separately, 50 micrograms of Lipofectin Reagent was diluted to 100 μlwith H₂O. The 100 μl of diluted Lipofectin Reagent was then addeddropwise to the diluted plasmid DNA contained in a polystyrene 5 ml snapcap tube and mixed gently. The mixture was then incubated for 15-20minutes at room temperature. During this time, the virus inoculum wasremoved from the 6 cm plates and the cell monolayers washed once withEMSK negative medium. Three ml of EMSK negative medium was then added tothe plasmid DNA/lipofectin mixture and the contents pipetted onto thecell monolayer. The cells were incubated overnight (about 16 hours) at37° C. in a humidified 5% CO₂ environment. The next day the 3 ml of EMSKnegative medium was removed and replaced with 5 ml EMSK complete medium.The cells were incubated at 37° C. in 5% CO₂ for 3-7 days untilcytopathic effect from the virus was 80-100%. Virus was harvested asdescribed above for the preparation of virus stocks. This stock wasreferred to as a transfection stock and was subsequently screened forrecombinant virus by the BLUOGAL SCREEN FOR RECOMBINANT SWINEPOX VIRUSOR CPRG SCREEN FOR RECOMBINANT SWINEPOX VIRUS.

SCREEN FOR RECOMBINANT SPV EXPRESSING β-GALACTOSIDASE (BLUOGAL AND CPRGASSAYS)

When the E. coli β-galactosidase (lacZ) marker gene was incorporatedinto a recombinant virus the plaques containing the recombinants werevisualized by one of two simple methods. In the first method, thechemical Bluogal™ (Bethesda Research Labs) was incorporated (200 μg/ml)into the agarose overlay during the plaque assay, and plaques expressingactive β-galactosidase turned blue. The blue plaques were then pickedonto fresh cells (EMSK) and purified by further blue plaque isolation.In the second method, CPFG (Boehringer Mannheim) was incorporated (400μg/ml) into the agarose overlay during the plaque assay, and plaquesexpressing active β-galactosidase turned red. The red plaques were thenpicked onto fresh cells (EMSK) and purified by further red plaqueisolation. In both cases viruses were typically purified with threerounds of plaque purification.

SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV USING BLACK PLAQUEASSAYS

To analyze expression of foreign antigens expressed by recombinantswinepox viruses, monolayers of EMSK cells were infected withrecombinant SPV, overlayed with nutrient agarose media and incubated for6-7 days at 37° C. for plaque development to occur. The agarose overlaywas then removed from the dish, the cells fixed with 100% methanol for10 minutes at room temperature and the cells air dried. Fixation of thecells results in cytoplasmic antigen as well as surface antigendetection whereas specific surface antigen expression can be detectedusing non-fixed cells. The primary antibody was then diluted to theappropriate dilution with PBS and incubated on the cell monolayer for 2hours at room temperature. To detect PRV g50 (gD) expression fromS-SPV-008, swine anti-PRV serum (Shope strain; lot370, PDV8201, NVSL,Ames, Iowa) was used (diluted 1:100). To detect NDV HN expression fromS-SPV-009, a rabbit antiserum specific for the HN protein (rabbitanti-NDV#2) was used (diluted 1:1000). Unbound antibody wag then removedby washing the cells three times with PBS at room temperature. Thesecondary antibody, either a goat anti-swine (PRV g50 (gD); S-SPV-008)or goat anti-rabbit (NDV HN; S-SPV-009), horseradish peroxidaseconjugate was diluted 1:250 with PBS and incubated with the cells for 2hours at room temperature. Unbound secondary antibody was then removedby washing the cells three times with PBS at room temperature. The cellswere then incubated 15-30 minutes at room temperature with freshlyprepared substrate solution (100 μg/ml 4-chloro-1-naphthol, 0.003% H₂O₂in PBS). Plaques expressing the correct antigen stain black.

PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS

Viral glycoproteins are purified using antibody affinity columns. Toproduce monoclonal antibodies, 8 to 10 week old BALB/c female mice arevaccinated intraperitoneally seven times at two to four week intervalswith 10⁷ PFU of S-SPV-009, -014, -016, -017, -018, or -019. Three weeksafter the last vaccination, mice are injected intraperitoneally with 40mg of the corresponding viral glycoprotein. Spleens are removed from themice three days after the last antigen dose.

Splenocytes are fused with mouse NS1/Ag4 plasmacytoma cells by theprocedure modified from oi and Herzenberg, (41). Splenocytes andplasmacytoma cells are pelleted together by centrifugation at 300×g for10 minutes. One ml of a 50% solution of polyethylene glycol (m.w.1300-1600) is added to the cell pellet with stirring over one minute.Dulbecco's modified Eagles's medium (5 ml) is added to the cells overthree minutes. Cells are pelleted by centrifugation at 300×g for 10minutes and resuspended in medium with 10% fetal bovine serum andcontaining 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine(HAT). Cells (100 ml) are added to the wells of eight to ten 96-welltissue culture plates containing 100 ml of normal spleen feeder layercells and incubated at 37° C. Cells are fed with fresh HAT medium everythree to four days.

Hybridoma culture supernatants are tested by the ELISA ASSAY in 96-wellmicrotiter plates coated with 100 ng of viral glycoprotein. Supernatantsfrom reactive hybridomas are further analyzed by black-plaque assay andby Western Blot. Selected hybridomas are cloned twice by limitingdilution. Ascetic fluid is produced by intraperitoneal injection of5×10⁶ hybridoma cells into pristane-treated BALB/c mice.

Cell lysates from S-SPV-009, -014, -016, -017, -018, or -019 areobtained as described in PREPARATION OF INFECTED CELL LYSATES. Theglycoprotein-containing cell lysates (100 mls) are passed through a 2-mlagarose affinity resin to which 20 mg of glycoprotein monoclonalantibody has been immobilized according to manufacturer's instructions(AFC Medium, New Brunswick Scientific, Edison, N.J.). The column iswashed with 100 ml of 0.1% Nonidet P-40 in phosphate-buffered saline(PBS) to remove nonspecifically bound material. Bound glycoprotein iseluted with 100 mM carbonate buffer, pH 10.6 (40). Pre- and postelutedfractions are monitored for purity by reactivity to the SPV monoclonalantibodies in an ELISA system.

ELISA ASSAY

A standard enzyme-linked immunosorbent assay (ELISA) protocol is used todetermine the immune status of cattle following vaccination andchallenge.

A glycoprotein antigen solution (100 ml at ng/ml in PBS) is allowed toabsorb to the wells of microtiter dishes for 18 hours at 4° C. Thecoated wellg are rinsed one time with PBS. Wells are blocked by adding250 ml of PBS containing 1% BSA (Sigma) and incubating 1 hour at 37° C.

The blocked wells are rinsed one time with PBS containing 0.02% Tween20. 50 ml of test serum (previously diluted 1:2 in PBS containing 1%BSA) are added to the wells and incubated 1 hour at 37° C. The antiserumis removed and the wells are washed 3 times with PBS containing 0.02%Tween 20. 50 ml of a solution containing anti-bovine IgG coupled tohorseradish peroxidase (diluted 1:500 in PBS containing 1% BSA,Kirkegaard and Perry Laboratories, Inc.) is added to visualize the wellscontaining antibody against the specific antigen. The solution isincubated 1 hour at 37° C., then removed and the wells are washed 3times with PBS containing 0.02% Tween 20. 100 ml of substrate solution(ATBS, Kirkegaard and Perry Laboratories, Inc.) are added to each welland color is allowed to develop for 15 minutes. The reaction isterminated by addition of 0.1M oxalic acid. The color is read atabsorbance 410 nm on an automatic plate reader.

STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC POX VIRAL PROMOTERS

For recombinant swinepox vectors synthetic pox promoters offer severaladvantages including the ability to control the strength and timing offoreign gene expression. Three promoter cassettes LP1, EP1 and LP2 basedon promoters that have been defined in the vaccinia virus (1, 7 and 8)were designed. Each cassette was designed to contain the DNA sequencesdefined in vaccinia flanked by restriction sites which could be used tocombine the cassettes in any order or combination. Initiator methionineswere also designed into each cassette such that inframe fusions could bemade at either EcoRI or BamHI sites. A set of translational stop codonsin all three reading frames and an early transcriptional terminationsignal (9) were also engineered downstream of the inframe fusion site.DNA encoding each cassette was synthesized according to standardtechniques and cloned into the appropriate homology vectors (see FIGS.4, 5 and 8).

VACCINATION STUDIES IN SWINE USING RECOMBINANT SWINEPOX VIRUS CONTAININGPSEUDORABIES VIRUS GLYCOPROTEIN GENES

Young weaned pigs from pseudorabies-free herd are used to test theefficacy of the recombinant swinepox virus containing one or more of thepseudorabies virus glycoprotein genes (SPV/PRV). The piglets areinoculated intramuscularly, intradermally or orally about 10³ to 10⁷plaque forming units (PFU) of the recombinant SPV/PRV viruses.

Immunity is determined by measuring PRV serum antibody levels and bychallenging the vaccinated pigs with virulent strain of pseudorabiesvirus. Three to four weeks post-vaccination, both vaccinated andnon-vaccinated groups of pigs are challenged with virulent strain ofpseudorabies virus (VDL4892). Post challenge, the pigs are observeddaily for 14 days for clinical signs of pseudorabies.

Serum samples are obtained at the time of vaccination, challenge, and atweekly intervals for two to three weeks post-vaccination and assayed forserum neutralizing antibody.

CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININ AND NEURAMINIDASE GENES

The equine influenza virus hemagglutinin (HA) and Neuraminidase (NA)genes was cloned essentially as described by Katz et al. (42) for the HAgene of human influenza virus. Viral RNA was prepared from virus grownin MDBK cells (for Influenza A/equine/Alaska/91 and InfluenzaA/equine/Miami/63) and MDCK cells (for Influenza A/equine/Prague/56 andInfluenza A/equine/Kentucky/81) was first converted to cDNA utilizing anoligo nucleotide primer specific for the target gene. The cDNA was usedas a template for PCR cloning (51) of the targeted gene region. The PCRprimers were designed to incorporate restriction sites which permit thecloning of the amplified coding regions into vectors containing theappropriate signals for expression in EHV. One pair of oligo nucleotideprimers was required for each coding region. The HA gene coding regionsfrom the serotype 2 (H3) viruses (Influenza A/equine/Miami/63, InfluenzaA/equine/Kentucky/81, and Influenza A/equine/Alaska/91) was clonedutilizing the following primers5′-GGAGGCCTTCATGACAGACAACCATTATTTTGATACTACTGA-3′ (SEQ ID NO: 120) forcDNA priming and combined with5′-GAAGGCCTTCTCAAATGCAAATGTTGCATCTGATGTTGCC-3′ (SEQ ID NO: 121) for PCR.The HA gene coding region from the serotype 1 (H7) virus (InfluenzaA/equine/Prague/56) was be cloned utilizing the following primers5′-GGGATCCATGAACACTCAAATTCTAATATTAG-3′. (SEQ ID NO: 122) for cDNApriming and combined with 5′-GGGATCCTTATATACAAATAGTGCACCGCA-3′ (SEQ IDNO: 123) for PCR. The NA gene coding regions from the serotype 2 (N8)viruses (Influenza A/equine/Miami/63, Influenza A/equine/Kentucky/81,and Influenza A/equine/Alaska/91) was cloned utilizing the followingprimers 5′-GGGTCGACATGAATCCAAATCAAAAGATAA-3′ (SEQ ID NO: 124) for cDNApriming and combined with 5′-GGGTCGACTTACATCTTATCGATGTCAAA-3′ (SEQ IDNO: 125) for PCR. The NA gene coding region from the serotype 1 (N7)virus (Influenza/A/equine/Prague/56) was cloned utilizing the followingprimers 5′-GGGATCCATGAATCCTAATCAAAAACTCTTT-3′ (SEQ ID NO: 118) for cDNApriming and combined with 5′-GGGATCCTTACGAAAAGTATTTAATTTGTGC-3′ (SEQ IDNO: 119) for PCR. Note that this general strategy was used to clone thecoding regions of HA and NA genes from other strains of equine influenzaA virus. The EIV HA or NA genes were cloned as a blunt ended SalI orBamHI fragment into a blunt ended EcoRI site behind the LP2EP2 promoterof the SPV homology vector.

CLONING OF PARAINFLUENZA-3 VIRUS FUSION AND HEMAGGLUTININ GENES

The parainfluenza-3 virus fusion (F) and hemagglutinin (HN) genes werecloned by a PCR CLONING procedure essentially as described by Katz etal. (42) for the HA gene of human influenza. Viral RNA prepared frombovine PI-3 virus grown in Madin-Darby bovine kidney (MDBK) cells wasfirst converted to cDNA utilizing an oligonucleotide primer specific forthe target gene. The cDNA was then used as a template for polymerasechain reaction (PCR) cloning (15) of the targeted region. The PCRprimers were designed to incorporate restriction sites which permit thecloning of the amplified coding regions into vectors containing theappropriate signals for expression in SPV. One pair of oligonucleotideswere required for each coding region. The F gene coding region from thePI-3 strain SF-4 (VR-281) was cloned using the following primers:5′-TTATGGATCCTGCTGCTGTGTTGAACAACTTTGT-3′ (SEQ ID NO: 130) for cDNApriming and combined with 5′-CCGCGGATCCCATGACCATCACAACCATAATCATAGCC-3′(SEQ ID NO: 131) for PCR. The HN gene coding region from PI-3 strainSF-4 (VR-281) was cloned utilizing the following primers:5′-CGTCGGATCCCTTAGCTGCAGTTTTTTGGAACTTCTGTTTTGA-3′ (SEQ ID NO: 132) forcDNA priming and combined with5′-CATAGGATCccATCAATATTGGAAACACACAAACAGCAC-3′ (SEQ ID NO: 133) for PCR.Note that this general strategy is used to clone the coding region of Fand HN genes from other strains of PI-3. The DNA fragment for PI-3 HN orF was digested with BamHI to yield an 1730 bp or 1620 bp fragment,respectively. The PI-3 HN fragment is cloned into the BamHI site next tothe LP2EP2 promoter of the SPV homology vector. The PI-3 F fragment wascloned into the BamHI site next to the LP2EP2 promoter of the SPVhomology vector to yield homology vector 713-55.10.

CLONING OF BOVINE VIRAL DIARRHEA VIRUS g48 AND g53 GENES

The bovine viral diarrhea g48 and g53 genes were cloned by a PCR CLONINGprocedure essentially as described by Katz et al. (42) for the HA geneof human influenza. Viral RNA prepared from BVD virus Singer straingrown in Madin-Darby bovine kidney (MDBK) cells was first converted tocDNA utilizing an oligonucleotide primer specific for the target gene.The cDNA was then used as a template for polymerase chain reaction (PCR)cloning (15) of the targeted region. The PCR primers were designed toincorporate restriction sites which permit the cloning of the amplifiedcoding regions into vectors containing the appropriate signals forexpression in SPV. One pair of oligonucleotides were required for eachcoding region. The g48 gene coding region from the BVDV Singer strain(49) was cloned using the following primers:5′-ACGTCGGATCCCTTACCAAACCACGTCTTACTCTTGTTTTCC-3′ (SEQ ID NO: 134) forcDNA priming and combined with5′-ACATAGGATCCCATGGGAGAAAACATAACACAGTGGAACC-3′ (SEQ ID NO: 135) for PCR.The g53 gene coding region from the BVDV Singer strain (49) was clonedusing the following primers: 5′-CGTGGATCCTCAATTACAAGAGGTATCGTCTAC-3′(SEQ ID NO: 136) for cDNA priming and combined with5′-CATAGATCTTGTGGTGCTGTCCGACTTCGCA-3′ (SEQ ID NO: 137) for PCR. Notethat this general strategy is used to clone the coding region of g48 andg53 genes from other strains of BVDV. The DNA fragment for BVDV g48 wasdigested with BamHI to yield an 678 bp fragment. The DNA fragment forBVDV g53 was digested with BglII and BamHI to yield an 1187 bp fragment.The BVDV g48 or g53 DNA fragments were cloned into the BamHI site nextto the LP2EP2 promoter of the SPV homology vector to yield homologyvectors, 727-78.1 and 738-96, respectively.

CLONING OF BOVINE RESPIRATORY SYNCYTIAL VIRUS FUSION, NUCLEOCAPSID ANDGLYCOPROTEIN GENES

The bovine respiratory syncytial virus fusion (F), nucleocapsid (N), andglycoprotein (G) genes were cloned by a PCR CLONING procedureessentially as described by Katz et al. (42) for the HA gene of humaninfluenza. Viral RNA prepared from BRSV virus grown in bovine nasalturbinate (BT) cells was first converted to cDNA utilizing anoligonucleotide primer specific for the target gene. The cDNA was thenused as a template for polymerase chain reaction (PCR) cloning (15) ofthe targeted region. The PCR primers were designed to incorporaterestriction sites which permit the cloning of the amplified codingregions into vectors containing the appropriate signals for expressionin SPV. One pair of oligonucleotides were required for each codingregion. The F gene coding region from the BRSV strain 375 (VR-1339) wascloned using the following primers:5′-TGCAGGATCCTCATTTACTAAAGGAAAGATTGTTGAT-3′ (SEQ ID NO: 138) for cDNApriming and combined with 5′-CTCTGGATCCTACAGCCATGAGGATGATCATCAGC-3′ (SEQID NO: 139) for PCR. The N gene coding region from BRSV strain 375(VR-1339) was cloned utilizing the following primers:5′-CGTCGGATCCCTCACAGTTCCACATCATTGTCTTTGGGAT-3′(SEQ ID NO: 140) for cDNApriming and combined with5′-CTTAGGATCCCATGGCTCTTAGCAAGGTCAAACTAAATGAC-3′ (SEQ ID NO: 141) forPCR. The G gene coding region from BRSV strain 375 (VR-1339) was clonedutilizing the following primers:5′-CGTTGGATCCCTAGATCTGTGTAGTTGATTGATTTGTGTGA-3′ (SEQ ID NO: 142) forcDNA priming and combined with5′-CTCTGGATCCTCATACCCATCATCTTAAATTCAAGACATTA-3′ SEQ ID NO: 143) for PCR.Note that this general strategy is used to clone the coding region of F,N and G genes from other strains of BRSV. The DNA fragments for BRSV F,N, or G were digested with BamHI to yield 1722 bp, 1173 bp, or 771 bpfragments, respectively. The BRSV F, N, and G DNA fragments were clonedinto the BamHI site next to the LP2EP2 promoter of the SPV homologyvector to yield homology vectors, 727-20.10, 713-55.37 and 727-20.5,respectively.

RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEEN CELLS

Chicken spleens were dissected from 3 week old SPAFAS hatched chicks,washed, and disrupted through a syringe/needle to release cells. Afterallowing stroma and debri to settle out, the cells were pelleted andwashed twice with PBS. The cell pellet was treated with a hypotoniclysis buffer to lyre red blood cells, and splenocytes were recovered andwashed twice with PBS. Splenocytes were resuspended at 5×10⁶ cells/ml inRPMI containing 5% FBS and 5 μg/ml Concanavalin A and incubated at 39°C. for 48 hours. Total RNA was isolated from the cells using guanidineisothionate lysis reagents and protocols from the Promega RNA isolationkit (Promega Corporation, Madison Wis.). 4 μg of total RNA was used ineach 1st strand reaction containing the appropriate antisense primersand AMV reverse transcriptase (Promega Corporation, Madison Wis.). cDNAsynthesis was performed in the same tube following the reversetranscriptase reaction, using the appropriate sense primers and Vent®DNA polymerase (Life Technologies, Inc. Bethesda, Md.).

HOMOLOGY VECTOR 515-85.1

The plasmid 515-85.1 was constructed for the purpose of insertingforeign DNA into SPV. It contains a unique AccI restriction enzyme siteinto which foreign DNA may be inserted. When a plasmid, containing aforeign DNA insert at the AccI site, is used according to the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a viruscontaining the foreign DNA will result. A restriction map of the DNAinsert in homology vector 515-85.1 is given in FIGS. 4A-4D. It may beconstructed utilizing standard recombinant DNA techniques (22 and 29),by joining two restriction fragments from the following sources. Thefirst fragment is an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). The second fragment is anapproximately 3628 base pair HindIII to BglII restriction sub-fragmentof the SPV HindIII restriction fragment M (23).

HOMOLOGY VECTOR 520-17.5

The plasmid 520-17.5 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by SPV DNA. Upstream of the marker gene is anapproximately 2149 base pair fragment of SPV DNA. Downstream of themarker gene is an approximately 1484 base pair fragment of SPV DNA. Whenthis plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV a virus containing DNA coding for themarker gene will result. Note that the β-galactosidase (lacZ) markergene is under the control of a synthetic early/late pox promoter. Adetailed description of the plasmid is given in FIGS. 4A-4D. It may beconstructed utilizing standard recombinant DNA techniques (22 and 30),by joining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 4A-4D. The plasmid vector wasderived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately2149 base pair HindIII to AccI restriction sub-fragment of the SPVHindIII restriction fragment M (23). Fragment 2 is an approximately 3006base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 3 is an approximately 1484 base pair AccI to BglII restrictionsub-fragment of the SPV HindIII fragment M (23).

HOMOLOGY VECTOR 538-46.16

The plasmid 538-46.16 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the PRV g50 (gD) gene flanked by SPV DNA. Upstream ofthe foreign genes is an approximately 2149 base pair fragment of SPVDNA. Downstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1) and the g50 (gD) gene is under thecontrol of a synthetic early/late pox promoter (EP1LP2). A detaileddescription of the plasmid is given in FIGS. 5A-5D. It may beconstructed utilizing standard recombinant DNA techniques (22 and 30),by joining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 5A-5D. The plasmid vector wasderived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately2149 base pair HindIII to AccI restriction sub-fragment of the SPVHindIII restriction fragment M (23). Fragment 2 is an approximately 3006base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 3 is an approximately 1571 base pair EcoRI to StuI restrictionsub-fragment of the PRV BamHI fragment 7 (21). Note that the EcoRI sitewas introduced in to this fragment by PCR cloning. In this procedure theprimers described below were used along with a template consisting of aPRV BamHI #7 fragment subcloned into pSP64. The first primer 87.03(5′-CGCGAATTCGCTCGCAGCGCTATTGGC-3′) (SEQ ID NO:41) sits down on the PRVg50 (gD) sequence (26) at approximately amino acid 3 priming toward the3′ end of the gene. The second primer 87.06 (5′-GTAGGAGTGGCTGCTGAAG-3′)(SEQ ID NO:42) sits down on the opposite strand at approximately aminoacid 174 priming toward the 5′ end of the gene. The PCR product may bedigested with EcoRI and SalI to produce an approximately 509 base pairfragment. The approximately 1049 base pair SalI to StuI sub-fragment ofPRV BamHI #7 may then be ligated to the approximately 509 base pairEcoRI to SalI fragment to generate the approximately 1558 base pairEcoRI to StuI fragment 3. Fragment 4 is an approximately 1484 base pairAccI to BglII restriction sub-fragment of the SPV HindIII fragment M(23).

HOMOLOGY VECTOR 570-91.21

The plasmid 570-91.21 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream ofthe foreign DNA genes is an approximately 1484 base pair fragment of SPVDNA. Downstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the gIII (gC) gene is under thecontrol of a synthetic early pox promoter (EP2). A detailed descriptionof the plasmid is given in FIGS. 10A-10D. It may be constructedutilizing standard recombinant DNA techniques (22 and 30), by joiningrestriction fragments from the following sources with the synthetic DNAsequences indicated in FIGS. 10A-10D. The plasmid vector was derivedfrom an approximately 2972 base pair HindIII to BamHI restrictionfragment of pSP64 (Promega). Fragment 1 is an approximately 1484 basepair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 3002 basepair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment ofplasmid 251-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkershave replaced the NcoI and NcoI sites at the ends of this fragment.Fragment 4 is an approximately 2149 base pair AccI to HindII restrictionsub-fragment of the SPV HindIII fragment M (23). The AccI sites infragments 1 and 4 have been converted to PstI sites using synthetic DNAlinkers.

HOMOLOGY VECTOR 570-91.41

The plasmid 570-91.41 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream ofthe foreign DNA genes is an approximately 2149 base pair fragment of SPVDNA. Downstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the gIII (gC) gene is under thecontrol of a synthetic early late pox promoter (EP1LP2). A detaileddescription of the plasmid is given in FIGS. 11A-11D. It may beconstructed utilizing standard recombinant DNA techniques (22 and 30),by joining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 11A-11D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction, sub-fragment of the SPVHindIII restriction fragment M (23). Fragment 2 is an approximately 3002base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment ofplasmid 251-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkershave replaced the NcoI and NcoI sites at the ends of this fragment.Fragment 4 is an approximately 2149 base pair AccI to HindIIIrestriction sub-fragment of the SPV HindIII fragment M (23). The AccIsites in fragments 1 and 4 have been converted to PstI sites usingsynthetic DNA linkers.

HOMOLOGY VECTOR 570-91.64

The plasmid 570-91.64 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the PRV gIII (gC) gene flanked by SPV DNA. Upstream ofthe foreign DNA genes is an approximately 1484 base pair fragment of SPVDNA. Downstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the gIII (gC) gene is under thecontrol of a synthetic late early pox promoter (LP2EP2). A detaileddescription of the plasmid is given in FIGS. 12A-12D. It may beconstructed utilizing standard recombinant DNA techniques (22 and 30),by joining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 12A-12D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 3002 basepair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 3 is an approximately 2378 base pair NcoI to NcoI fragment ofplasmid 251-41.A, a subfragment of PRV BamHI #2 and #9. EcoRI linkershave replaced the NcoI and NcoI sites at the ends of this fragment.Fragment 4 is an approximately 2149 base pair AccI to HindIIIrestriction sub-fragment of the SPV HindIII fragment M (23). The AccIsites in fragments 1 and 4 have been converted to PstI sites usingsynthetic DNA linkers.

HOMOLOGY VECTOR 538-46.26

The plasmid 538-46.26 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the Newcastle Disease Virus (NDV)hemagglutinin-Neuraminidase (HN) gene flanked by SPV DNA.

Upstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. Downstream of the foreign genes is an approximately1484 base pair fragment of SPV DNA. When this plasmid is used accordingto the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPVa virus containing DNA coding for the foreign genes will result. Notethat the β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1) and the HN gene is under the controlof a synthetic early/late pox promoter (EP1LP2). A detailed descriptionof the plasmid is given in FIGS. 8A-8D. It may be constructed utilizingstandard recombinant DNA techniques (22 and 30), by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated in FIGS. 8A-8D. The plasmid vector was derived from anapproximately 2972 base pair HindIII to BamHI restriction fragment ofpSP64 (Promega). Fragment 1 is an approximately 2149 base pair HindIIIto AccI restriction sub-fragment of the SPV HindIII restriction fragmentM (23). Fragment 2 is an approximately 1810 base pair AvaII to NaeIrestriction fragment of a NDV HN cDNA clone. The sequence of the HN cDNAclone is given in FIG. 7. The cDNA clone was generated from the B1strain of NDV using standard cDNA cloning techniques (14). Fragment 3 isan approximately 3006 base pair BamHI to PvuII restriction fragment ofplasmid pJF751 (11). Fragment 4 is an approximately 1484 base pair AccIto BglII restriction sub-fragment of the SPV HindIII fragment M (23).

HOMOLOGY VECTOR 599-65.25

The plasmid 599-65.25 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the ILT gG gene flanked by SPV DNA. Upstream of theforeign genes is an approximately 1484 base pair fragment of SPV DNA.Downstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the ILT gG gene is under thecontrol of a synthetic early/late pox promoter (EP1LP2). A detaileddescription of the plasmid is given in FIGS. 13A-13D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 13A-13D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 1073 basepair EcoRI to MboI fragment. Note that the EcoRI site was introduced byPCR cloning. In this procedure, the primers described below were usedwith a template consisting of a 2.6 kb Sst I to Asp718I subfragment of a5.1 kb Asp718I fragment of ILT virus genome. The first primer 91.13(5′-CCGAATTCCGGCTTCAGTAACATAGGATCG-3′) (SEQ ID NO: 81) sits down on theILT gG sequence at amino acid 2. It adds an additional asparagineresidue between amino acids 1 and 2 and also introduces an EcoRIrestriction site. The second primer 91.14 (5′-GTACCCATACTGGTCGTGGC-3′)(SEQ ID NO: 82) sits down on the opposite strand at approximately aminoacid 196 priming toward the 5′ end of the gene. The PCR product isdigested with EcoRI and BamHI to produce an approximately 454 base pairfragment. The approximately 485 base pair MboI sub-fragment of ILTAsp718I (5.1 kb) fragment is ligated to the approximately 454 base pairEcoRI to BamHI fragment to generate fragment 2 from EcoRI to MboI whichis approximately 939 base pairs (293 amino acids) in length. Fragment 3is an approximately 3002 base pair BamHlI to PvuII restriction fragmentof plasmid pJF751 (11). Fragment 4 is an approximately 2149 base pairACCI to HindIII subfragment of the SPV HindIII fragment M. The AccIsites of fragments 1 and 4 have been converted to PstI sites usingsynthetic DNA linkers.

HOMOLOGY VECTOR 624-20.1C

The plasmid 624-20.1C was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacz)marker gene and the ILT gI gene flanked by SPV DNA. Upstream of theforeign genes is an approximately 1484 base pair fragment of SPV DNA.Downstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the ILT gI gene is under thecontrol of a synthetic late/early pox promoter (LP2EP2). A detaileddescription of the plasmid is given in FIGS. 14A-14D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 14A-14D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 1090 basepair fragment with EcoRI and BamHI restriction sites at the endssynthesized by PCR cloning and containing the entire amino acid codingsequence of the ILT gI gene. The ILT gI gene was synthesized in twoseparate PCR reactions. In this procedure, the primers described belowwere used with a template consisting the 8.0 kb ILT Asp 718I fragment.The first primer 103.6 (5′-CCGGAATTCGCTACTT GGAACTCTGG-3′) (SEQ ID NO:83) sits down on the ILT gI sequence at amino acid number 2 andintroduces an EcoRI site at the 5′ end of the ILT gI gene. The secondprimer 103.3 (5′-CATTGTCCCGAGACGGACAG-3′) (SEQ ID NO: 84) sits down onthe ILT gI sequence at approximately amino acid 269 on the oppositestrand to primer 103.6 and primes toward the 5′ end of the gene. The PCRproduct was digested with EcoRI and BglI (BglI is located approximatelyat amino acid 209 which is 179 base pairs 5′ to primer 2) to yield afragment 625 base pairs in length corresponding to the 5′ end of the ILTgI gene. The third primer 103.4 (5′-CGCGATCCAACTATCGGTG-3′) (SEQ ID NO:85) sits down on the ILT gI gene at approximately amino acid 153 primingtoward the 3′ end of the gene. The fourth primer 103.5(5′GCGGATCCACATTCAG ACTTAATCAC-3′) (SEQ ID NO: 86) sits down at the 3′end of the ILT gI gene 14 base pairs beyond the UGA stop codon,introducing a BamHI restriction site and priming toward the 5′ end ofthe gene. The PCR product is digested with Bgl I (at amino acid 209) andBamHI to yield a fragment 476 base pairs in length corresponding to the3′ end of the ILT gI gene. Fragment 2 consists of the products of thetwo PCR reactions ligated together to yield an ILT gI gene which is aEcoRI to BamHI fragment approximately 1101 base pairs (361 amino acids)in length. Fragment 3 is an approximately 3002 base pair BamHI to PvuIIrestriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 2149 base pair AccI to HindIII subfragment of the SPVHindIII fragment M. The AccI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 614-83.18

The plasmid 614-83.18 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the IBR gG gene flanked by SPV DNA. Upstream of theforeign genes is an approximately 1484 base pair fragment of SPV DNA.Downstream of the foreign genes is an approximately 2149 base pairfragment of SPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter (LP1), and the IBR gG gene is under thecontrol of a synthetic late/early pox promoter (LP2EP2). A detaileddescription of the plasmid is given in FIGS. 15A-15D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 15A-15D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 1085 basepair fragment synthesized by PCR cloning with EcoRI and BamHIrestriction sites at the ends and containing the amino acid codingsequence from amino acids 2 to 362 of the IBR gG gene. In the PCRcloning procedure, the primers described below were used with a templateconsisting of the IBR-000 virus (Cooper strain). The first primer 106.9(5′-ATGAATTCCCCTGCCGCCCGGACCGGCACC-3′) (SEQ ID NO: 87) sits down on theIBR gG sequence at amino acid number 1 and introduces an EcoRI site atthe 5′ end of the IBR gG gene and two additional amino acids betweenamino acids 1 and 2. The second primer 106.8(5′-CATGGATCCCGCTCGAGGCGAGCGGGCTCC-3′) (SEQ ID NO: 88) sits down on theIBR gG sequence at approximately amino acid 362 on the opposite strandto primer 1 and primes synthesis toward the 5′ end of the IBR gG gene.Fragment 2 was generated by digesting the PCR product with EcoRI andBamHI to yield a fragment 1085 base pairs in length corresponding to theamino terminal 362 amino acids (approximately 80%) of the IBR gG gene.Fragment 3 is an approximately 3002 base pair BamHI to PvuII restrictionfragment of plasmid pJF751 (11). Fragment 4 is an approximately 2149base pair AccI to HindIII subfragment of the SPV HindIII fragment M. TheAccI sites in fragments 1 and 4 were converted to unique NotI sitesusing NotI linkers.

HOMOLOGY VECTOR FOR CONSTRUCTING S-SPV-019 (LacZ/IBR gE HOMOLOGY VECTOR)

This lacZ/IBR gE homology vector is used to insert foreign DNA into SPV.It incorporates an E. coli β-galactosidase (lacZ) marker gene and theIBR gE gene flanked by SPV DNA. When this plasmid is used according tothe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of asynthetic late pox promoter and the gE gene is under the control of asynthetic late/early pox promoter. The homology vector may beconstructed utilizing standard. recombinant DNA techniques (22 and 30),by joining restriction fragments from the following sources with theappropriate synthetic DNA sequences. The plasmid vector is derived froman approximately 2972 base pair HindIII to BamHI restriction fragment ofpSP64 (Promega). The upstream SPV homology is an approximately 1146 basepair BglIII to AccI restriction sub-fragment of the SPV HindIII fragmentM (23). The IBR gE gene is an approximately 1888 base pair fragmentsynthesized by PCR cloning with EcoRI and BamHI ends. In the PCR cloningprocedure, the primers described below were used with a templateconsisting of the IBR-000 VIRUS (Cooper strain). The first primer4/93.17DR (5′-CTGGTTCGGCCCAGAATTCTATGGGTCTCGCGCGGCTCGTGG-3′ (SEQ ID NO:89) sits down on the IBR gE gene at amino acid number 1 and introducesan EcoRI site at the 5′ end of the IBR gE gene and adds two additionalamino acids at the amino terminus of the protein. The second primer4/93.18DR (5′-CTCGCTCGCCCAGGATCCCTAGCGGAGGATGGACTTGAGTCG-3′) (SEQ ID NO:90) sits down on the IBR gE sequence at approximately amino acid 648 onthe opposite strand to primer 1 and primes synthesis toward the 5′ endof the IBR gE gene. The lacZ promoter and marker gene is identical tothe one used in plasmid 520-17.5. The downstream SPV homology is anapproximately 2156 base pair AccI to HindIII restriction sub-fragment ofthe SPV HindIII restriction fragment M (23). The AccI site in the SPVhomology vector is converted to a unique XbaI site.

HOMOLOGY VECTOR FOR CONSTRUCTING S-SPV-018 (LacZ/PRV gE HOMOLOGY VECTOR)

This homology vector is constructed for the purpose of inserting foreignDNA into SPV. It incorporates an E. coli β-galactosidase (lacZ) markergene and the PRV gE gene flanked by SPV DNA. Upstream of the foreigngenes is an approximately 1484 base pair fragment of SPV DNA. Downstreamof the foreign genes is an approximately 2149 base pair fragment of SPVDNA. When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing the DNAcoding for the foreign genes results. Note that the β-galactosidase(lacZ) marker gene is under the control of a synthetic late pox promoter(LP1), and the PRV gE gene is under the control of a syntheticearly/late pox promoter (EP1LP2). The homology vector is constructedutilizing standard recombinant DNA techniques (22,30), by joiningrestriction fragments from the following sources with synthetic DNAsequences. The plasmid vector is derived from an approximately 2972 basepair HindIII to BamHI restriction fragment pSP64 (Promega). Fragment 1is an approximately 1484 base pair BglII to AccI restrictionsub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2is the lacZ promoter and marker gene which is identical to the one usedin plasmid 520-17.5. Fragment 3 is an approximately 2484 base pair DraIto MluI sub-fragment of PRV derived from the PRV BamHI #7 DNA fragment.The DraI site is converted to an EcoRI site through the use of asynthetic DNA linker. The DraI site sits 45 base pairs upstream of thenatural gE start codon and extends the open reading frame at the aminoterminus of the protein for 15 amino acids. The synthetic poxpromoter/EcoRI DNA linker contributes another 4 amino acids. Therefore,the engineered gE gene contains 19 additional amino acids fused to theamino terminus of gE. The nineteen amino acids areMet-Asn-Ser-Gly-Asn-Leu-Gly-Thr-Pro-Ala-Ser-Leu-Ala-His-Thr-Gly-Val-Glu-Thr.Fragment 4 is an approximately 2149 base pair AccI to HindIIIsub-fragment of the SPV HindIII fragment M (23). The AccI sites offragments 1 and 4 are converted to PstI sites using synthetic DNAlinkers.

HOMOLOGY VECTOR 520-90.15

The plasmid 520-90.15 was constructed for the purpose of insertingforeign DNA into SPV. It contains a unique NdeI restriction enzyme siteinto which foreign DNA may be inserted. When a plasmid, containing aforeign DNA insert at the NdeI site, is used according to the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV a viruscontaining the foreign DNA will result. Plasmid 520-90.15 wasconstructed utilizing standard recombinant DNA techniques (22 and 30),by joining two restriction fragments from the following sources. Thefirst fragment is an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). The second fragment is anapproximately 1700 base pair HindIII to BamHI restriction subfragment ofthe SPV HindIII restriction fragment G (23).

HOMOLOGY VECTOR 708-78.9

The plasmid 708-78.9 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the infectious bovine rhinotracheitis virus (IBRV) gEgene flanked by SPV DNA. Upstream of the foreign genes is anapproximately 1484 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 2149 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a synthetic late pox promoter (LP1),and the IBRV gE gene is under the control of a synthetic late/early poxpromoter (LP2EP2). It may be constructed utilizing standard recombinantDNA techniques (22, 30), by joining restriction fragments from thefollowing sources. The plasmid vector was derived from an approximately2972 base pair HindIII to BamHI restriction fragment of pSP64 (Promega).Fragment 1 is an approximately 1484 base pair BglII to AccI restrictionsub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2is an approximately 475 base pair fragment with EcoRI and BamHIrestriction sites at the ends. The EcoRI and BamHI sites are synthesizedby PCR cloning. The PCR product contains the entire amino acid codingsequence of the IBRV gE gene. In the PCR cloning procedure, the primersdescribed below were used with a template consisting of the IBR-000virus (Cooper strain) (44). The first primer 2/94.5DR(5′-CTGGTTCGGCCCAGAATTCGATGCAACCCACCGCGCCGCCCCG-3′) (SEQ ID NO: 116)sits down on the IBR gE gene at amino acid number 1 and introduces anEcoRI site at the 5′ end of the IBRV gE gene and adds two additionalamino acids at the amino terminus of the protein. The second primer4/93.18DR (5′-CTCGCTCGCCCAGGATCCCTAGCGGAGGATGGACTTGAGTCG-3′) (SEQ ID NO:117) sits down on the IBRV gE sequence (44) at approximately amino acid648 on the opposite strand to the first primer and primes synthesistoward the 5′ end of the IBRV gE gene. The PCR product was digested withEcoRI and BamHI to yield a fragment approximately 1950 base pairs inlength corresponding to the IBRV gE gene. Fragment 3 is an approximately3002 base pair BamHI to PvuII restriction fragment of plasmid pJF75l(11). Fragment 4 is an approximately 2149 base pair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccI sites in fragments 1 and4 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 723-59A9.22

The plasmid 723-59A9.22 was used to insert foreign DNA into SPV. Itincorporates an E. coli β-galactosidase (lacZ) marker gene and theequine influenza virus NA PR/56 gene flanked by SPV DNA. When thisplasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT SPV a virus containing DNA coding for the foreigngenes results. Note that the β-galactosidase (lacZ) marker gene is underthe control of a synthetic late pox promoter (LP1) and the EIV PR/56 NAgene is under the control of a synthetic late/early pox promoter(LP2EP2). A detailed description of the plasmid is given in FIGS.18A-18D. The homology vector was constructed utilizing standardrecombinant DNA techniques (22 and 30), by joining restriction fragmentsfrom the following sources with the appropriate synthetic DNA sequences.The plasmid vector was derived from an approximately 2972 base pairHindIII to BamHI restriction fragment of pSP64 (Promega). Fragment 1 isan approximately 1484 base pair BglII to AccI restriction sub-fragmentof the SPV HindIII fragment M (23). Fragment 2 is the NA gene codingregion from the equine Influenza A/Prague/56 (serotype 1 (N7) virus)cloned as an approximately 1450 base pair BamHI fragment utilizing thefollowing primers 5-GGGATCCATGAATCCTAATCAAAAACTCTTT-3′ (SEQ ID NO: 118)for cDNA priming and combined with 5′-GGGATCCTTACGAAAAGTATTTAATTTGTGC-3′(SEQ ID NO: 119) for PCR. (see CLONING OF EQUINE INFLUENZA VIRUSHEMAGGLUTININ AND NEURAMINIDASE GENES). Fragment 3 is an approximately3010 base pair BamHI to PvuII restriction fragment of plasmid pJF751(11). Fragment 4 is an approximately 2149 base pair AccI to HindIIIrestriction sub-fragment of the SPV HindIII restriction fragment M (23).The AccI site in the SPV homology vector was converted to a unique NotIsite.

HOMOLOGY VECTOR 727-54.60

The plasmid 727-54.60 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the pseudorabies virus (PRV) gII (gB) gene flanked bySPV DNA. Upstream of the foreign genes is an approximately 1484 basepair fragment of SPV DNA. Downstream of the foreign genes is anapproximately 2149 base pair fragment of SPV DNA. When the plasmid isused according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV, a virus containing DNA coding for the foreign geneswill result. Note that the β-galactosidase (lacZ) marker gene is underthe control of a synthetic late pox promoter (LP1), and the PRV gB geneis under the control of a synthetic late/early pox promoter (LP2EP2). Adetailed description of the plasmid is given in FIGS. 19A-19D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 19A-19D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 3500 basepair fragment which contains the coding sequence for the PRV gB genewithin the KpnI C fragment of genomic PRV DNA (21). Fragment 2 containsan approximately 53 base air synthetic fragment containing the aminoterminus of the PRV gB gene, an approximately 78 base pair SmaI to Nhe Ifragment from the PRV KpnI C genomic fragment, and an approximately 3370base pair NheI to EcoRI fragment from the PRV KpnI C genomic fragment(21). Fragment 3 is an approximately 3010 base pair BamHI to PvuIIrestriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 2149 base pair AccI to HindIII subfragment of the SPVHindIII fragment M. The AccI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 727-67.18

The plasmid 727-67.18 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the hepatitis B virus core antigen gene flanked by SPVDNA. Upstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. Downstream of the foreign genes is an approximately2149 base pair fragment of SPV DNA. When the plasmid is used accordingto the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTSPV, a virus containing DNA coding for the foreign genes will result.Note that the β-galactosidase (lacZ) marker gene is under the control ofa synthetic late pox promoter (LP1), and the hepatitis B core antigengene is under the control of a synthetic early/late pox promoter(EP1LP2). A detailed description of the plasmid is given in FIGS.20A-20D. It may be constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources with the synthetic DNA sequences indicated in FIGS. 20A-20D. Theplasmid vector was derived from an approximately 2972 base pair HindIIIto BamHI restriction fragment of pSP64 (Promega). Fragment 1 is anapproximately 1484 base pair BglII to AccI restriction sub-fragment ofthe SPV HindIII restriction fragment M (23). Fragment 2 is anapproximately 3002 base air BamHI to PvuII restriction fragment ofplasmid pJF751 (11). Fragment 3 is an approximately 589 base pairfragment with BamHI and EcoRI restriction sites at the ends. Thisfragment contains the hepatitis B core antigen coding sequences (aminoacids 25-212) (Ref. 45, 50). Fragment 4 is an approximately 2149 basepair AccI to HindIII subfragment of the SPV HindIII fragment M. The AccIsites in fragments 1 and 4 were converted to unique NotI sites usingNotI linkers.

HOMOLOGY VECTOR 732-18.4

The plasmid 732-18.4 was used to insert foreign DNA into SPV. Itincorporates an E. coli β-galactosidase (lacZ) marker gene and theequine influenza virus AK/91 NA gene flanked by SPV DNA. When thisplasmid was used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FORGENERATING RECOMBINANT SPV a virus containing DNA coding for the foreigngenes results. Note that the β-galactosidase (lacz) marker gene is underthe control of a synthetic late pox promoter (LP1) and the EIV AK/91 NAgene is under the control of a synthetic late/early pox promoter(LP2EP2). A detail description of the plasmid is given in FIGS. 21A-21D.The homology vector was constructed utilizing standard recombinant DNAtechniques (22 and 30), by joining restriction fragments from thefollowing sources with the appropriate synthetic DNA sequences. Theplasmid vector was derived from an approximately 2972 base pair HindIIIto BamHI restriction fragment of pSP64 (Promega). Fragment 1 is anapproximately 1484 base pair BglII to AccI restriction sub-fragment ofthe SPV HindIII fragment M (23). Fragment 2 is the NA gene coding regionfrom the equine Influenza A/Alaska/91 (serotype 2 (N8) virus) cloned asan approximately 1450 base pair SalI fragment utilizing the followingprimers 5′-GGGTCGACATGAATCCAAATCAAAAGATAA-3′ (SEQ ID NO: 124) for cDNApriming and combined with 5′-GGGTCGACTTACATCTTATCGATGTCAAA-3′ (SEQ IDNO: 125) for PCR (see CLONING OF EQUINE INFLUENZA VIRUS HEMAGGLUTININAND NEURAMINIDASE GENES). Fragment 3 is an approximately 3010 base pairBamHI to PvuII restriction fragment of plasmid pJF751 (11). Fragment 4is an approximately 2149 base pair AccI to HindIII restrictionsub-fragment of the SPV HindIII restriction fragment M (23). The AccIsite in the SPV homology vector was converted to a unique NotI site

HOMOLOGY VECTOR 741-80.3

The plasmid 741-80.3 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. Coli β-galactosidase (lacZ)marker gene flanked by SPV DNA. Upstream of the foreign genes is anapproximately 1484 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 2149 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a human cytomegalovirus immediateearly (HCMV IE) promoter. A detailed description of the plasmid is givenin FIGS. 22A-22C. It may be constructed utilizing standard recombinantDNA techniques (22, 30), by joining restriction fragments from thefollowing sources with the synthetic DNA sequences indicated in FIGS.22A-22C. The plasmid vector was derived from an approximately 2972 basepair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment1 is an approximately 1484 base pair BglII to AccI restrictionsub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2is a 1154 base pair PstI to AvaII fragment derived from a HCMV 2.1 kbPstI fragment containing the HCMV IE promoter (46). Fragment 3 is a 3010base pair BamHI to PvuII fragment derived from plasmid pJF751 (49)containing the E. coli lacZ gene. Fragment 4 is an approximately 750base pair NdeI to SalI fragment derived from PRV BamHI #7 which containsthe carboxy-terminal 19 amino acids and the polyadenylation signal ofthe PRV gX gene. Fragment 5 is an approximately 2149 base pair AccI toHindIII subfragment of the SPV HindIII fragment M. The AccI sites infragments 1 and 5 were converted to unique NotI sites using NotIlinkers.

HOMOLOGY VECTOR 741-84.14

The plasmid 741-84.14 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the human interleukin-2 (IL-2) gene flanked by SPV DNA.Upstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. Downstream of the foreign genes is an approximately2149 base pair fragment of SPV DNA. When the plasmid is used accordingto the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTSPV, a virus containing DNA coding for the foreign genes will result.Note that the β-galactosidase (lacZ) marker gene is under the control ofa synthetic late pox promoter (LP1), and the human IL-2 gene is underthe control of a synthetic late/early pox promoter (LP2EP2). The codingsequence for the human IL-2 protein is fused at the amino terminus tothe PRV gX signal sequence for membrane transport. A detaileddescription of the plasmid is given in FIGS. 23A-23D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 23A-23D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 475 basepair fragment with EcoRI and BglII restriction sites at the ends. TheEcoRI site is synthesized by PCR cloning and the BglII site is at the 3′end of the human IL-2 cDNA (43, 47). The PCR product contains the entireamino acid coding sequence of the PRV gX signal sequence-human IL-2gene. In this procedure, the primers described below were used with atemplate consisting of the cDNA for PRV gX signal sequence-human IL-2(43). The first primer 5/94.23 (5′-CTCGAATTCGAAGTGGGCAACGTGGATCCTCGC-3′)(SEQ ID NO: 126) sits down on the PRV gX signal sequence at amino acidnumber 1 and introduces an EcoRI site at the 5′ end of the gene. Thesecond primer 5/94.24 (5′-CAGTTAGCCTCCCCCATCTCCCCA-3′) (SEQ ID NO: 127)sits down on the human IL-2 gene sequence within the 3′ untranslatedregion on the opposite strand to primer 5/94.23 and primes toward the 5′end of the gene. The PCR product was digested with EcoRI and BglII(BglII is located approximately 3 nucleotides beyond the stop codon forthe human IL-2 gene) to yield a fragment 475 base pairs in lengthcorresponding to the PRV gX signal sequence-human IL-2 gene. Fragment 3is an approximately 3010 base pair BamHI to PvuII restriction fragmentof plasmid pJF751 (11). Fragment 4 is an approximately 2149 base pairAccI to HindIII subfragment of the SPV HindIII fragment M. The AccIsites in fragments 1 and 4 were converted to unique NotI sites usingNotI linkers.

HOMOLOGY VECTOR 744-34

The plasmid 744-34 was constructed for the purpose of inserting foreignDNA into SPV. It incorporates an E. coli β-galactosidase (lacZ) markergene and the equine herpesvirus type 1 gB gene flanked by SPV DNA.Upstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. Downstream of the foreign genes is an approximately2149 base pair fragment of SPV DNA. When the plasmid is used accordingto the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTSPV, a virus containing DNA coding for the foreign genes will result.Note that the β-galactosidase (lacZ) marker gene is under the control ofa synthetic late pox promoter (LP1), and the EHV-1 gB gene is under thecontrol of a synthetic late/early pox promoter (LP2EP2). A detaileddescription of the plasmid is given in FIGS. 24A-24D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 24A-24D The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 2941 basepair fragment with EcoRI and PmeI restriction sites at the ends.Fragment 2 is an approximately 2941 base pair EcoRI to PmeI fragment.Fragment 2 was synthesized as an approximately 429 base pair PCRfragment at the 5′ end of the gene having a synthetic EcoRI site and anatural BamHI site within the BamHI “a” fragment of EHV-1 genomic DNAand an approximately 2512 base pair restriction fragment at the 3′ endof the gene from BamHI to PmeI within the BamHI “i” fragment of EHV-1genomic DNA (48). In the procedure to produce the 5′ end PCR fragment,the primers described below were used with a template consisting of theEHV-1 BamHI “a” and “i” fragments. The first primer 5/94.3(5′-CGGAATTCCTCTGGTTGCCGT-3′) (SEQ ID NO: 128) sits down on the EHV-1 gBsequence at amino acid number 2 and introduces an EcoRI site at the 5′end of the EHV-1 gB gene and an ATG start codon. The second primer5/94.4 (5′-GACGGTGGATCCGGTAGGCGGT-3′) (SEQ ID NO: 129) sits down on theEHV-1 gB sequence at approximately amino acid 144 on the opposite strandto primer 5/94.3 and primes toward the 5′ end of the gene. The PCRproduct was digested with EcoRI and BamHI to yield a fragment 429 basepairs in length corresponding to the 5′ end of the EHV-1 gB gene.Fragment 2 consists of the products of the PCR reaction (EcoRI to BamHI)and the restriction ragment (BamHI to PmeI) ligated together to yield anEHV-1 gB gene which is an EcoRI to PmeI fragment approximately 2941 basepairs (979 amino acids) in length. Fragment 3 is an approximately 3010base pair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 4 is an approximately 2149 base pair AccI to HindIIIsubfragment of the SPV HindIII fragment M. The AccI sites in fragments 1and 4 were converted to unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 744-38

The plasmid 744-38 was constructed for the purpose of inserting foreignDNA into SPV. It incorporates an E. coli β-galactosidase (lacZ) markergene and the equine herpesvirus type 1 gD gene flanked by SPV DNA.Upstream of the foreign genes is an approximately 1484 base pairfragment of SPV DNA. Downstream of the foreign genes is an approximately2149 base pair fragment of SPV DNA. When the plasmid is used accordingto the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANTSPV, a virus containing DNA coding for the foreign genes will result.Note that the β-galactosidase (lacZ) marker gene is under the control ofa synthetic late pox promoter (LP1), and the EHV-1 gD gene is under thecontrol of a synthetic late/early pox promoter (LP2EP2). A detaileddescription of the plasmid is given in FIGS. 25A-25D. It may beconstructed utilizing standard recombinant DNA techniques (22, 30), byjoining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 25A-25D. The plasmid vectorwas derived from an approximately 2972 base pair HindIII to BamHIrestriction fragment of pSP64 (Promega). Fragment 1 is an approximately1484 base pair BglII to AccI restriction sub-fragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 1240 basepair HindIII fragment within the BamHI “d” fragment of EHV-1 (48).Fragment 3 is an approximately 3010 base pair BamHI to PvuII restrictionfragment of plasmid pJF751 (11). Fragment 4 is an approximately 2149base pair AccI to HindIII subfragment of the SPV HindIII fragment M. TheAccI sites in fragments 1 and 4 were converted to unique NotI sitesusing NotI linkers.

HOMOLOGY VECTOR 689-50.4

The plasmid 689-50.4 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the infectious bursal disease virus (IBDV) polyproteingene flanked by SPV DNA. Upstream of the foreign genes is anapproximately 1484 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 2149 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a synthetic late pox promoter (LP1),and the IBDV polyprotein gene is under the control of a syntheticlate/early pox promoter (LP2EP2). It may be constructed utilizingstandard recombinant DNA techniques (22, 30), by joining restrictionfragments from the following sources. The plasmid vector was derivedfrom an approximately 2972 base pair HindIII to BamHI restrictionfragment of pSP64 (Promega). Fragment 1 is an approximately 1484 basepair BglII to AccI restriction subfragment of the SPV HindIIIrestriction fragment M (23). Fragment 2 is an approximately 3400 basepair fragment with SmaI and HpaI restriction sites at the ends fromplasmid 2-84/2-40 (51). This fragment contains the IBDV polyproteincoding sequences. Fragment 3 is an approximately 3010 base pair BamHI toPvuII restriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 2149 base pair AccI to HindIII subfragment of the SPVHindIII fragment M. The AccI sites in fragments 1 and 4 were convertedto unique NotI sites using NotI linkers.

HOMOLOGY VECTOR 689-50.7

The plasmid 689-50.7 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and the infectious bursal disease virus (IBDV) VP2 geneflanked by SPV DNA. Upstream of the foreign genes is an approximately1484 base pair fragment of SPV DNA. Downstream of the foreign genes isan approximately 2149 base pair fragment of SPV DNA. When the plasmid isused according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV, a virus containing DNA coding for the foreign geneswill result. Note that the β-galactosidase (lacZ) marker gene is underthe control of a synthetic late pox promoter (LP1), and the IBDV VP2gene is under the control of a synthetic late/early pox promoter(LP2EP2). It may be constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources. The plasmid vector was derived from an approximately 2972 basepair HindIII to BamHI restriction fragment of pSP64 (Promega). Fragment1 is an approximately 1484 base pair BglII to AccI restrictionsub-fragment of the SPV HindIII restriction fragment M (23). Fragment 2is an approximately 1081 base pair fragment with BclI and BamHIrestriction sites at the ends. This fragment codes for the IBDV VP2protein and is derived from a full length IBDV cDNA clone (51). Fragment3 is an approximately 3010 base pair BamHI to PvuII restriction fragmentof plasmid pJF751 (11). Fragment 4 is an approximately 2149 base pairAccI to HindIII sub-fragment of the SPV HindIII fragment M. The AccIsites in fragments 1 and 4 were converted to unique NotI sites usingNotI linkers.

HOMOLOGY VECTOR 751-07.A1

The plasmid 751-07.A1 was used to insert foreign DNA into SPV. Itincorporates an E. coli β-galactosidase (lacz) marker gene and thechicken interferon (cIFN) gene flanked by SPV DNA. When this plasmid wasused according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV a virus containing DNA coding for the foreign genesresults. Note that the β-galactosidase (lacZ) marker gene is under thecontrol of a synthetic late pox promoter (LP1) and the cIFN gene isunder the control of a synthetic late/early pox promoter (LP2EP2). Thehomology vector was constructed utilizing standard recombinant DNAtechniques (22 and 30), by joining restriction fragments from thefollowing sources with the appropriate synthetic DNA sequences. Theplasmid vector was derived from an approximately 2972 base pair HindIIIto BamHI restriction fragment of pSP64 (Promega). Fragment 1 is anapproximately 1146 base pair BglII to AccI restriction sub-fragment ofthe SPV HindIII fragment M (23). Fragment 2 is an approximately 577 basepair EcoRI to BglII fragment coding for the cIFN gene (54) derived byreverse transcription and polymerase chain reaction (PCR) (Sambrook, etal., 1989) of RNA ISOLATED FROM CONCANAVALIN A STIMULATED CHICKEN SPLEENCELLS. The antisense primer (6/94.13) used for reverse transcription andPCR was 5′-CGACGGATCCGAGGTGCGTTTGGGGCTAAGTGC-3′ (SEQ ID NO: 211). Thesense primer (6/94.12) used for PCR was5′-CCACGGATCCAGCACAACGCGAGTCCCACCATGGCT-3′ (SEQ ID NO: 212). The BamHIfragment resulting from reverse transcription and PCR was gel purifiedand used as a template for a second PCR reaction to introduce a uniqueEcoRI site at the 5′ end and a unique BglII site at the 3′ end. Thesecond PCR reaction used primer 6/94.22(5′-CCACGAATTCGATGGCTGTGCCTGCAAGCCCACAG-3′; SEQ ID NO: 213) at the 5′end and primer 6/94.34 (5′-CGAAGATCTGAGGTGCGTTTGGGGCTAAGTGC-3′; SEQ IDNO: 214) at the 3′ end to yield an approximately 577 base pair fragment.The DNA fragment contains the coding sequence from amino acid 1 to aminoacid 193 of the chicken interferon protein (54) which includes a 31amino acid signal sequence at the amino terminus and 162 amino acids ofthe mature protein encoding chicken interferon. Fragment 3 is anapproximately 3002 base pair BamHI to PvuII restriction fragment ofplasmid pJF751 (11). Fragment 4 is an approximately 2156 base pair AccIto HindIII restriction sub-fragment of the SPV HindIII restrictionfragment M (23). The AccI site in the SPV homology vector wag convertedto a unique NotI site.

HOMOLOGY VECTOR 751-56.A1

The plasmid 751-56.A1 was used to insert foreign DNA into SPV. Itincorporates an E. coli β-galactosidase (lacZ) marker gene and thechicken myelomonocytic growth factor (cMGF) gene flanked by SPV DNA.When this plasmid was used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV a virus containing DNA codingfor the foreign genes results. Note that the β-galactosidase (lacZ)marker gene is under the control of a synthetic late pox promoter (LP1)and the cMGF gene is under the control of a synthetic late/early poxpromoter (LP2EP2). The homology vector was constructed utilizingstandard recombinant DNA techniques (22 and 30), by joining restrictionfragments from the following sources with the appropriate synthetic DNAsequences. The plasmid vector was derived from an approximately 2972base pair HindIII to BamHI restriction fragment of pSP64 (Promega).Fragment 1 is an approximately 1146 base pair BglII to AccI restrictionsub-fragment of the SPV HindIII fragment M (23). Fragment 2 is anapproximately 640 base pair EcoRI to BamHI fragment coding for the cMGFgene (55) derived by reverse transcription and polymerase chain reaction(PCR) (Sambrook, et al., 1989) of RNA ISOLATED FROM CONCANAVALIN ASTIMULATED CHICKEN SPLEEN CELLS. The antisense primer (6/94.20) used forreverse transcription and PCR was5′-CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3′ (SEQ ID NO: 215). The senseprimer (5/94.5) used for PCR was 5′-GAGCGGATCCTGCAGGAGGAGACACAGAGCTG-3′(SEQ ID NO: 216). The BamHI fragment derived from PCR was subcloned intoa plasmid and used as a template for a second PCR reaction using primer6/94.16 (5′-GCGCGAATTCCATGTGCTGCCTCACCCCTGTG-3′; SEQ ID NO: 217) at the5′ end and primer 6/94.20 (5′-CGCAGGATCCGGGGCGTCAGAGGCGGGCGAGGTG-3′; SEQID NO: 218) at the 3′ end to yield an approximately 640 base pairfragment. The DNA fragment contains the coding sequence from amino acid1 to amino acid 201 of the cMGF protein (55) which includes a 23 aminoacid signal sequence at the amino terminus and 178 amino acids of themature protein encoding cMGF. Fragment 3 is an approximately 3002 basepair BamHI to PvuII restriction fragment of plasmid pJF751 (11).Fragment 4 is an approximately 2156 base pair AccI to HindIIIrestriction sub-fragment of the SPV HindIII restriction fragment M (23).The AccI site in the SPV homology vector was converted to a unique NotIsite.

HOMOLOGY VECTOR 752-22.1

The plasmid 752-22.1 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene flanked by SPV DNA. Upstream of the foreign gene is anapproximately 855 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 1113 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a swinepox virus O1L gene promoter.The homology vector also contains the synthetic late/early promoter(LP2EP2) into which a second foreign gene is inserted into a uniqueBamHI or EcoRI site. A detailed description of the plasmid is given inFIGS. 28A-28D. It was constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources with the synthetic DNA sequences indicated in FIGS. 28A-28D. Theplasmid vector was derived from an approximately 2519 base pair HindIIIto SphI restriction fragment of pSP65 (Promega). Fragment 1 is anapproximately 855 base pair sub-fragment of the SPV HindIII restrictionfragment M (23) synthesized by polymerase chain reaction using DNAprimers 5′-GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3′ (SEQ ID NO: 185)and 5′-CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG-3′, (SEQ ID NO: 186) toproduce an 855 base pair fragment with SphI and BglII ends. Fragment 2is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751(49) containing the E. coli lacZ gene. Fragment 3 is an approximately1113 base pair subfragment of the SPV HindIII fragment M synthesized bypolymerase chain reaction using DNA primers5′-CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3′ (SEQ ID NO: 187) and5′-GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3′, (SEQ ID NO: 188) toproduce an 1113 base pair fragment with SalI and HindIII ends.

HOMOLOGY VECTOR 752-29.33

The plasmid 759.33 was constructed for the purpose of inserting foreignDNA into SPV. It incorporates an E. coli β-galactosidase (lacZ) markergene and an equine herpesvirus type 1 gB gene flanked by SPV DNA.Upstream of the foreign gene is an approximately 855 base pair fragmentof SPV DNA. Downstream of the foreign genes is an approximately 113 basepair fragment of SPV DNA. When the plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV, avirus containing DNA coding for the foreign genes will result. Note thatthe β-galactosidase (lacZ) marker gene is under the control of aswinepox virus 01L gene promoter and the EHV-1 gB gene is under thecontrol of the late/early promoter (LP2EP2). The LP2EP2 promoter-EHV-1gB gene cassette was inserted into a NotI site of homology vector738-94.4. Homology vector 752-29.33 was constructed utilizing standardrecombinant DNA techniques (22, 30), by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences. The plasmidvector was derived from an approximately 2519 base pair HindIII to SphIrestriction fragment of pSP65 (Promega). Fragment 1 is an approximately855 base pair sub-fragment of the SPV HindIII restriction fragment M(23) synthesized by polymerase chain reaction using DNA primers5′-GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3′ (SEQ ID NO: 185) and5′-CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG-3′ (SEQ ID NO: 186) toproduce an 855 base pair fragment with SphI and BglII ends. Fragment 2is a 3002 base pair BamHI to PvuII fragment derived from plasmid pJF751(49) containing the E. coli lacz gene. Fragment 3 is the product of aPCR reaction (EcoRI to BamHI) and a restriction fragment (BamHI to PmeI)ligated together to yield an EHV-1 gB gene which is an EcoRI to PmeIfragment approximately 2941 base pairs (979 amino acids) in length. ThePCR fragment is an approximately 429 base pair fragment having asynthetic EcoRI site at the 5′ end of the gene and a natural BamHI siteat the 3′ end within the BamHI “a” fragment of EHV-1 genomic DNA. Therestriction fragment is an approximately 2512 base pair fragment fromBamHI to PmeI within the BamHI “I” fragment. of EHV-1 genomic DNA. Inthe procedure to produce the 5′ end PCR fragment, the primers describedbelow were used with a template consisting of the EHV-1 BamHI “a” and“i” fragments.

The first primer 5/94.3 (5′-CGGAATTCCTCTGGTTCGCCGT-3′) (SEQ ID NO: 128)sits down on the EHV-1 gB sequence at amino acid number 2 and introducesan EcoRI site at the 5′ end of the EHV-1 gB gene and an ATG start codon.The second primer 5/94.4 (5′-GACGGTGGATCCGGTAGGCGGT-3′) (SEQ ID NO: 129)sits down on the EHV-1 gB sequence at approximately amino acid 144 onthe opposite strand to primer 5/94.3 and primes toward the 5′ end of thegene. The PCR product was digested with EcoRI and BamHI to yield afragment 429 base pairs in length corresponding to the 5′ end of theEHV-1 gB gene. Fragment 3 consists of the products of the PcR reaction(EcoRI to BamHI) and the restriction fragment (BamHI to PmeI) ligatedtogether to yield an EHV-1 gB gene which is an EcoRI to PmeI fragmentapproximately 2941 base pairs (979 amino acids) in length. Fragment 4 isan approximately 1113 base pair subfragment of the SPV HindIII fragmentM synthesized by polymerase chain reaction using DNA primers5′-CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3′ (SEQ ID NO: 187) and5′-GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3′ (SEQ ID NO: 188) toproduce an 1113 base pair fragment with SalI and HindIII ends.

HOMOLOGY VECTOR 746-94.1

The plasmid 746-94.1 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and an infectious bovine rhinotracheitis virus glycoproteinE (gE) gene flanked by SPV DNA. Upstream of the foreign gene is anapproximately 855 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 1113 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a swinepox virus O1L gene promoterand the IBRV gE gene is under the control of the late/early promoter(LP2EP2). It was constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources with the synthetic DNA sequences. A 1250 base pair EcoRI toBamHI fragment coding for amino acids 1 to 417 of the IBRV gE gene(missing 158 amino acids of the carboxy terminal transmembrane region)was inserted into unique EcoRI and BamHI sites of homology vector752-22.1 (FIGS. 28A-28D). The 1250 base pair EcoRI to BamHI fragment wassynthesized by polymerase chain reaction (15) using IBRV (Cooper)genomic DNA as a template and primer 10/94.23(5′-GGGGAATTCAATGCAACCCACCGCGCCGCCCC-3′; (SEQ ID NO: 219) at the 5′ endof the IBRV gE gene (amino acid 1) and primer 10/94.22(5′-GGGGGATCCTAGGGCGCGCCCGCCGGCTCGCT-3′; SEQ ID NO: 220) at amino acid417 of the IBRV gE gene.

HOMOLOGY VECTOR 767-67.3

The plasmid 767-67.3 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and an bovine viral diarrhea virus glycoprotein 53 (BVDVgp53) gene flanked by SPV DNA. Upstream of the foreign gene is anapproximately 855 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 1113 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacZ)marker gene is under the control of a swinepox virus O1L gene promoterand the BVDV gp53 gene is under the control of the late/early promoter(LP2EP2). It was constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources with the synthetic DNA sequences. A 1187 base pair BamHIfragment coding for the BVDV gp53 was inserted into the unique BamHIsites of homology vector 752-22.1 (FIGS. 28A-28D). The 1187 base pairBamHI fragment was synthesized by polymerase chain reaction (15) asdescribed in CLONING OF BOVINE VIRAL DIARRHEA VIRUS gp48 AND gp53 GENES.

HOMOLOGY VECTOR 771-55.11

The plasmid 771-55.11 was constructed for the purpose of insertingforeign DNA into SPV. It incorporates an E. coli β-galactosidase (lacZ)marker gene and an bovine viral diarrhea virus glycoprotein 48 (BVDVgp48) gene flanked by SPV DNA. Upstream of the foreign gene is anapproximately 855 base pair fragment of SPV DNA. Downstream of theforeign genes is an approximately 1113 base pair fragment of SPV DNA.When the plasmid is used according to the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV, a virus containing DNA codingfor the foreign genes will result. Note that the β-galactosidase (lacz)marker gene is under the control of a swinepox virus O1L gene promoterand the BVDV gp48 gene is under the control of the late/early promoter(LP2EP2). It was constructed utilizing standard recombinant DNAtechniques (22, 30), by joining restriction fragments from the followingsources with the synthetic DNA sequences. A 678 base pair BamHI fragmentcoding for the BVDV gp48 was inserted into the unique BamHI sites ofhomology vector 752-22.1 (FIGS. 28A-28D). The 678 base pair BamHIfragment was synthesized by polymerase chain reaction (15) as describedin CLONING OF BOVINE VIRAL DIARRHEA VIRUS gp48 AND gp53 GENES.

EXAMPLES Example 1 Homology Vector 515-85.1

The homology vector 515-85.1 is a plasmid useful for the insertion offoreign DNA into SPV. Plasmid 515-85.1 contains a unique AccIrestriction site into which foreign DNA may be cloned. A plasmidcontaining such a foreign DNA insert may be used according to theHOMOLOCOUG RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV togenerate a SPV containing the foreign DNA. For this procedure to besuccessful it is important that the insertion site (AccI) be in a regionnon-essential to the replication of the SPV and that the site be flankedwith swinepox virus DNA appropriate for mediating homologousrecombination between virus and plasmid DNAs. AccI site in homologyvector 515-85.1 is used to insert foreign DNA into at least threerecombinant SPV (see examples 2-4).

In order to define an appropriate insertion site, a library of SPVHindIII restriction fragments was generated. Several of theserestriction fragments (HindIII fragments G, J, and M see FIGS. 1A-1B)were subjected to restriction mapping analysis. Two restriction siteswere identified in each fragment as potential insertion sites. Thesesites included HpaI and NruI in fragment G, BalI and XbaI in fragment J,and AccI and PstI in fragment M. A β-galactosidase (lacZ) marker genewas inserted in each of the potential sites. The resulting plasmids wereutilized in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV. The generation of recombinant virus was determined bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-GALACTOSIDASE ASSAYS. Fourof the six sites were found to generate recombinant virus, however theability of each of these viruses to be purified away from the parentalSPV varied greatly. In one case virus could not be purified above thelevel of 1%, in another case virus could not be purified above the levelof 50%, and in a third case virus could not be purified above the levelof 90%. The inability to purify these viruses indicates instability atthe insertion site. This makes the corresponding sites inappropriate forinsertion of foreign DNA. However the insertion at one site, the AccIsite of Homology vector 515-85.1, resulted in a virus which was easilypurified to 100% (see example 2), clearly defining an appropriate sitefor the insertion of foreign DNA.

The homology vector 515-85.1 was further characterize by DNA sequenceanalysis. Two regions of the homology vector were sequenced. The firstregion covers a 599 base pair sequence which flanks the unique AccI site(see FIGS. 2A and 2B). The second region covers the 899 base pairsupstream of the unique HindIII site (see FIGS. 2A and 2B). The sequenceof the first region codes for an open reading frame (ORF) which showshomology to amino acids 1 to 115 of the vaccinia virus (VV) O1L openreading frame identified by Goebel et al, 1990 (see FIGS. 3A-3C). Thesequence of the second region codes for an open reading frame whichshows homology to amino acids 568 to 666 of the same vaccinia virus O1Lopen reading frame (see FIGS. 3A-3C). These data suggest that the AccIsite interrupts the presumptive VV O1L-like ORF at approximately aminoacid 41, suggesting that this ORF codes for a gene non-essential for SPVreplication. Goebel et al. suggest that the VV O1L ORF contains aleucine zipper motif characteristic of certain eukaryotictranscriptional regulatory proteins, however they indicate that it isnot known whether this gene is essential for virus replication.

The DNA sequence located upstream of the VV 01L-like ORF (see FIG. 2A)would be expected to contain a swinepox viral promoter. This swinepoxviral promoter will be useful as the control element of foreign DNAintroduced into the swinepox genome.

Example 2 S-SPV-003

S-SPV-003 is a swinepox virus that expresses a foreign gene. The genefor E. coli β-galactosidase (lacZ gene) was inserted into the SPV515-85.1 ORF. The foreign gene (lacZ) is under the control of asynthetic early/late promoter (EP1LP2).

S-SPV-003 was derived from S-SPV-001 (Kasza strain). This wasaccomplished utilizing the homology vector 520-17.5 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-GALACTOSIDASE (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-003. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable andexpressing the foreign gene. The assays described here were carried outin VERO cells as well as EMSK cells, indicating that VERO cells would bea suitable substrate for the production of SPV recombinant vaccines.S-SPV-003 has been deposited with the ATCC under Accession No. VR 2335.

Example 3 S-SPV-008

S-SPV-008 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ gene) and the gene forpseudorabies virus (PRV) g50 (gD) (26) were inserted into the SPV515-85.1 ORF. The lacz gene is under the control of a synthetic latepromoter (LP1) and the g50 (gD) gene is under the control of a syntheticearly/late promoter (EP1LP2).

S-SPV-008 was derived from S-SPV-001 (Kasza strain). This wasaccomplished utilizing the homology vector 538-46.16 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-GALACTOSIDASE (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-008. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable andexpressing the marker gene.

S-SPV-008 was assayed for expression of PRV specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Swine anti-PRV serum was shown to react specifically with S-SPV-008plaques and not with S-SPV-009 negative control plaques. All S-SPV-008observed plaques reacted with the swine antiserum indicating that thevirus was stably expressing the PRV foreign gene. The black plaque assaywas also performed on unfixed monolayers. The SPV plaques on the unfixedmonolayers also exhibited specific reactivity with swine anti-PRV serumindicating that the PRV antigen is expressed on the infected cellsurface.

To confirm the expression of the PRV g50 (gD) gene product, cells wereinfected with SPV and samples of infected cell lysates were subjected toSDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzedusing the WESTERN BLOTTING PROCEDURE. The swine anti-PRV serum was usedto detect expression of PRV specific proteins. As shown in FIG. 6, thelysate from S-SPV-008 infected cells exhibits a specific band ofapproximately 48 kd, the reported size of PRV g50 (gD) (35).

PRV g50 (gD) is the g50 (gD) homologue of HSV-1 (26). Severalinvestigators have shown that VV expressing HSV-1 g50 (gD) will protectmice against challenge with HSV-1 (6 and 34). Therefore the S-SPV-008should be valuable as a vaccine to protect swine against PRV disease.

It is anticipated that several other PRV glycoproteins will be useful inthe creation of recombinant swinepox vaccines to protect against PRVdisease. These PRV glycoproteins include gII (28), gIII (27), and gH(19). The PRV gIII coding region has been engineered behind severalsynthetic pox promoters. The techniques utilized for the creation ofS-SPV-008 will be used to create recombinant swinepox viruses expressingall four of these PRV glycoprotein genes. Such recombinant swinepoxviruses will be useful as vaccines against PRV disease. Since the PRVvaccines described here do not express PRV gX or gI, they would becompatible with current PRV diagnostic tests (gX HerdChek®, gI HerdChek®and ClinEase®) which are utilized to distinguish vaccinated animals frominfected animals. S-SPV-008 has been deposited with the ATCC underAccession No. VR 2339.

Example 4 S-SPV-011

S-SPV-011 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacz) and the gene forpseudorabies virus gIII (gC) were inserted into the unique PstIrestriction site (PstI linkers inserted into a unique AccI site) of thehomology vector 570-33.32. The lac Z gene is under the control of thesynthetic late promoter (LP1) and the PRV gIII (gC) gene is under thecontrol of the synthetic early promoter (EP2).

S-SPV-011 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 570-91.21 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-011. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-011 was assayed for expression of PRV specific antigen using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Polyclonal goat anti-PRV gIII (gC) antibody was shown to reactspecifically with S-SPV-011 plaques and not with S-SPV-001 negativecontrol plaques. All S-SPV-011 observed plaques reacted with the swineanti-PRV serum indicating that the virus was stably expressing the PRVforeign gene. The assays described here were carried out in EMSK cells,indicating that EMSK cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the PRV gIII (gC) gene product, cells wereinfected with SPV and samples of infected cell lysates were subjected toSDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzedusing the WESTERN BLOTTING PROCEDURE. Polyclonal goat anti-PRV gIII (gC)antibody was used to detect expression of PRV specific proteins. Asshown in FIG. 16, the lysate from S-SPV-011 infected cells exhibits aspecific band of approximately 92 kd, the reported size of PRV gIII (gC)(37).

Recombinant-expressed PRV gIII (gC) has been shown to elicit asignificant immune response in mice and swine (37, 38). Furthermore,when gIII (gC) is coexpressed with gII (gB) or g50 (gD), significantprotection from challenge with virulent PRV is obtained (39). ThereforeS-SPV-011 should be valuable as a vaccine to protect swine against PRVdisease. Since the PRV vaccines described here do not express PRV gX orgI, they would be compatible with current PRV diagnostic tests (gXHerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguishvaccinated animals from infected animals.

Example 5 S-SPV-012

S-SPV-012 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene forpseudorabies virus gIII (gC) were inserted into the unique PstIrestriction site (PstI linkers inserted into a unique AccI site) of thehomology vector 570-33.32. The lacZ gene is under the control of thesynthetic late promoter (LP1) and the PRV gIII (gC) gene is under thecontrol of the synthetic early late promoter (EP1LP2).

S-SPV-012 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 570-91.41 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-012. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-012 was assayed for expression of PRV specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Polyclonal goat anti-PRV gIII (gC) antibody was shown to reactspecifically with S-SPV-012 plaques and not with S-SPV-001 negativecontrol plaques. All S-SPV-012 observed plaques reacted with the swineanti-PRV serum, indicating that the virus was stably expressing the PRVforeign gene. The assays described here were carried out in EMSK andVERO cells, indicating that EMSK cells would be a suitable substrate forthe production of SPV recombinant vaccines.

To confirm the expression of the PRV gIII (gC) gene product, cells wereinfected with S-SPV-012 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal goatanti-PRV gIII (gC) antibody was used to detect expression of PRVspecific proteins. As shown in FIG. 16, the lysate from S-SPV-012infected cells exhibits two specific bands which are the reported sizeof PRV gIII (gC) (37)—a 92 kd mature form and a 74 kd pre-golgi form.

Recombinant-expressed PRV gIII (gC) has been shown to elicit asignificant immune response in mice and swine (37, 38). Furthermore,when gIII (gC) is coexpressed with gII (gB) or g50 (gD), significantprotection from challenge with virulent PRV is obtained (39). ThereforeS-SPV-012 should be valuable as a vaccine to protect swine against PRVdisease. Since the PRV vaccines described here do not express PRV gX orgI, they would be compatible with current PRV diagnostic tests (gXHerdChek®, gI HerdChek® and ClinEase®) which are utilized to distinguishvaccinated animals from infected animals.

Example 6 S-SPV-013

S-SPV-013 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene forpseudorabies virus gIII (gC) were inserted into the unique PstIrestriction site (PstI linkers inserted into a unique AccI site) of thehomology vector 570-33.32. The lacZ gene is under the control of thesynthetic late promoter (LP1) and the PRV gIII (gC) gene is under thecontrol of the synthetic late early promoter (LP2EP2).

S-SPV-013 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 570-91.64 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-013. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-013 was assayed for expression of PRV specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Polyclonal goat anti-PRV gIII (gC) antibody was shown to reactspecifically with S-SPV-013 plaques and not with S-SPV-001 negativecontrol plaques. All S-SPV-013 observed plaques reacted with the swineanti-PRV serum indicating that the virus was stably expressing the PRVforeign gene. The assays described here were carried out in EMSK andVERO cells, indicating that EMSK cells would be a suitable substrate forthe production of SPV recombinant vaccines.

To confirm the expression of the PRV gIII (gC) gene product, cells wereinfected with SPV and samples of infected cell lysates were subjected toSDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzedusing the WESTERN BLOTTING PROCEDURE. Polyclonal goat anti-PRV gIII (gC)antibody was used to detect expression of PRV specific proteins. Asshown in FIG. 16, the lysate from S-SPV-013 infected cells exhibits twospecific bands which are the reported size of PRV gIII (gC) (37)—a 92 kdmature form and a 74 kd pre-Golgi form.

Recombinant-expressed PRV gIII (gC) has been shown to elicit asignificant immune response in mice and swine (37, 38). Furthermore,when gIII (gC) is coexpressed with gII (gB) or g50 (gD), significantprotection from challenge with virulent PRV is obtained. (39) ThereforeS-SPV-013 is valuable as a vaccine to protect swine against PRV disease.Since the PRV vaccines described here do not express PRV gX or gI, theywould be compatible with current PRV diagnostic tests (gX HerdChek®, gIHerdChek® and ClinEase®) which are utilized to distinguish vaccinatedanimals from infected animals. S-SPV-013 has been deposited with theATCC under Accession No. 2418.

Protection against Aujeszky's disease using recombinant swinepox virusvaccines S-SPV-008 and S-SPV-013.

A vaccine containing S-SPV-008 and S-SPV-013 (1×10⁶PFU/ml) (2 ml of a1:1 mixture of the two viruses) was given to two groups of pigs (5 pigsper group) by intradermal inoculation or by oral/pharyngeal spray. Acontrol group of 5 pigs received S-SPV-001 by both intradermal andoral/pharyngeal inoculation. Pigs were challenged three weekspost-vaccination with virulent PRV, strain 4892, by intranasalinoculation. The table presents a summary of clinical responses. Thedata support an increase in protection against Aujeszky's disease in theS-SPV-008/S-SPV-013 vaccinates compared to the S-SPV-001 vaccinatecontrols.

Post- Post- challenge challenge Post- Group Respira- challenge average:tory Signs: CNS signs: (Days of Route of (# with signs/ (# with signs/clinical Vaccine inoculation total number) total number) signs)S-SPV-008 + Intradermal 3/5 0/5 2.6 S-SPV-013 S-SPV-008 + Oral/ 3/5 0/52.2 S-SPV-013 pharyngeal S-SPV-001 Intra- 5/5 2/5 7.8 (Control) dermal +Oral/ Pharyngeal

Example 7 S-SPV-015

S-SPV-015 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene forpseudorabies virus (PRV) gII (gB) were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the PRV gB gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-015 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 727-54.60 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-015. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-015 was assayed for expression of PRV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Polyclonal swine anti-PRV serum was shown to react specifically withS-SPV-015 plaques and not with S-SPV-001 negative control plaques. AllS-SPV-015 observed plaques reacted with the antiserum indicating thatthe virus was stably expressing the PRV foreign gene. The assaysdescribed here were carried out in ESK-4 cells, indicating that ESK-4cells would be a suitable substrate for the production of SPVrecombinant vaccines.

To confirm the expression of the PRV gII gene product, cells wereinfected with SPV-015 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal swineanti-PRV serum was used to detect expression of PRV specific proteins.The lysate from S-SPV-015 infected cells exhibited bands correspondingto 120 kd, 67 kd and 58 kd, which are the expected size of the PRV gIIglycoprotein.

S-SPV-015 is useful as a vaccine in swine against pseudorabies virus. Asuperior vaccine is formulated by combining S-SPV-008 (PRV g50),S-SPV-013 (PRV gIII), and S-SPV-015 for protection against pseudorabiesin swine.

Therefore S-SPV-015 should be valuable as a vaccine to protect swineagainst PRV disease. Since the PRV vaccines described here do notexpress PRV gX or gI, they would be compatible with current PRVdiagnostic tests (gX HerdChek®, gI HerdChek® and ClinEase®) which areutilized to distinguish vaccinated animals from infected animals.S-SPV-015 has been deposited with the ATCC under Accession No. 2466.

Example 8

Recombinant swinepox virus expressing more than one pseudorabies virus(PRV) glycoproteins, which can elicit production of neutralizingantibodies against pseudorabies virus, is constructed in order to obtaina recombinant swinepox virus with enhanced ability to protect againstPRV infection than that which can be obtained by using a recombinantswinepox virus expressing only one of those PRV glycoproteins.

There are several examples of such recombinant swinepox virus expressingmore than one PRV glycoproteins: a recombinant swinepox virus expressingPRV g50 (gD) and gIII (gC), a recombinant swinepox virus expressing PRVg50 (gD) and gII (gB); a recombinant swinepox virus expressing PRV gII(gB) and gIII (gC); and a recombinant swinepox virus expressing PRV g50(gD), gIII (gC) and gII (gB). Each of the viruses cited above is alsoengineered to contain and express E. coli β-galactosidase (lacZ) gene,which will facilitate the cloning of the recombinant swinepox virus.

Listed below are three examples of a recombinant swinepox virusexpressing PRV g50 (gD), PRV gIII (gC), PRV gII (gB) and E. coliβ-galactosidase (lacZ):

a) Recombinant swinepox virus containing and expressing PRV g50 (gD)gene, PRV gIII (gC) gene, PRV gII (gB) gene and lacZ gene. All fourgenes are inserted into the unique AccI restriction endonuclease sitewithin the HindIII M fragment of the swinepox virus genome. PRV g50 (gD)gene is under the control of a synthetic early/late promoter (EP1LP2),PRV gIII (gC) gene is under the control of a synthetic early promoter(EP2), PRV gII (gB) gene is under the control of a synthetic late/earlypromoter (LP2EP2) and lacZ gene is under the control of a synthetic latepromoter (LP1).

b) Recombinant swinepox virus containing and expressing PRV g50 (gD)gene, PRV gIII (gC) gene, PRV gII (gB) gene and lacZ gene. All fourgenes are inserted into the unique AccI restriction endonuclease sitewithin the HindIII M fragment of the swinepox virus genome. PRV g50 (gD)gene is under the control of a synthetic early/late promoter (EP1LP2),PRV gIII (gC) gene is under the control of a synthetic early/latepromoter (EP1LP2), PRV gII (gB) gene is under the control of a syntheticlate/early promoter (LP2EP2) and lacZ gene is under the control of asynthetic late promoter (LP1).

c) Recombinant swinepox virus containing and expressing PRV g50 (gD)gene, PRV gIII (gC) gene, PRV gII (gB) gene and lacZ gene. All fourgenes are inserted into the unique AccI restriction endonuclease sitewithin the HindIII M fragment of the swinepox virus genome. PRV g50 (gD)gene is under the control of a synthetic early/late promoter (EP1LP2),PRV gIII (gC) gene is under the control of a synthetic late/earlypromoter (LP2EP2), PRV gII (gB) gene is under the control of a syntheticlate/early promoter (LP2EP2) and lacz gene is under the control of asynthetic late promoter (LP1).

Protection against Aujeszky's disease using recombinant swinepox virusvaccines S-SPV-008, S-SPV-013 and S-SPV-015.

A vaccine containing S-SPV-008, S-SPV-013, or S-SPV-015 (2 ml of 1×10⁷PFU/ml of the virus) or a mixture of S-SPV-008, S-SPV-013, and S-SPV-015(2 ml of a 1:1:1 mixture of the three viruses; 1×10⁷ PFU/ml) was givento four groups of pigs (5 pigs per group) by intramuscular inoculation.A control group of 5 pigs received S-SPV-001 by intramuscularinoculation. Pigs were challenged four weeks post-vaccination withvirulent PRV, strain 4892, by intranasal inoculation. The pigs wereobserved daily for 14 days for clinical signs of pseudorabies, and thetable presents a summary of clinical responses. The data show that pigsvaccinated with S-SPV-008, S-SPV-013, or S-SPV-015 had partialprotection and pigs vacinated with the combination vaccineS-SPV-008/S-SPV-013/S-SPV-015 had complete protection against Aujeszky'sdisease caused by pseudorabies virus compared to the S-SPV-001 vaccinatecontrols.

Post- Post- challenge challenge Post- Group Respira- challenge average:tory Signs: CNS signs: (Days of Route of (# with signs/ (# with signs/clinical Vaccine inoculation total number) total number) signs)S-SPV-008 Intra- 2/5 2/5 2.0 muscular S-SPV-013 Intra- 1/5 0/5 0.4muscular S-SPV-015 Intra- 3/5 0/5 1.0 muscular S-SPV-008 + Intra- 0/50/5 0.0 muscular S-SPV-013 + S-SPV-015 S-SPV-001 Intra- 5/5 2/5 3.6(Control) muscular

Example 9 S-SPV-009

S-SPV-009 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ gene) and the gene forNewcastle's Disease virus hemagglutinin (HN) gene were inserted into theSPV 515-85.1 ORF. The lacZ gene is under the control of a synthetic latepromoter (LP1) and the HN gene is under the control of an syntheticearly/late promoter (EP1LP2).

S-SPV-009 was derived from S-SPV-001 (Kasza strain). This wasaccomplished utilizing the homology vector 538-46.26 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-GALACTOSIDASE (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-009. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable andexpressing the marker gene.

S-SPV-009 was assayed for expression of PRV specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Rabbit anti-NDV HN serum was shown to react specifically with S-SPV-009plaques and not with S-SPV-008 negative control plaques. All S-SPV-009observed plaques reacted with the swine antiserum indicating that thevirus was stably expressing the NDV foreign gene. S-SPV-009 has beendeposited with the ATCC under Accession No. VR 2344).

To confirm the expression of the NDV HN gene product, cells wereinfected with SPV and samples of infected cell lysates were subjected toSDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzedusing the WESTERN BLOTTING PROCEDURE. The rabbit anti-NDV HN serum wasused to detect expression of the HN protein. The lysate from S-SPV-009infected cells exhibited a specific band of approximately 74 kd, thereported size of NDV HN (29).

Example 10 S-SPV-014

S-SPV-014 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiouslaryngotracheitis virus glycoprotein G (ILT gG) were inserted into theSPV 570-33.32 ORF (a unique PstI site has replaced the unique AccIsite). The lacZ gene is under the control of the synthetic late promoter(LP1), and the ILT gG gene is under the control of the syntheticearly/late promoter (EP1LP2).

S-SPV-014 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 599-65.25 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-014. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene. The assays described here were carried outin ESK-4 cells, indicating that ESK-4 cells would be a suitablesubstrate for the production of SPV recombinant vaccines.

To confirm the expression of the ILT gG gene product, cells wereinfected with SPV-014 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Peptide antisera toILT gG was used to detect expression of ILT specific proteins. Thelysate from S-SPV-014 infected cells exhibited a band at 43 kd which isthe expected size of the ILT gG protein and additional bands of highermolecular weight which represent glycosylated forms of the protein whichare absent in deletion mutants for ILT gG.

This virus is used as an expression vector for expressing ILTglycoprotein G (gG). Such ILT gG is used as an antigen to identifyantibodies directed against the wild-type ILT virus as opposed toantibodies directed against gG deleted ILT viruses. This virus is alsoused as an antigen for the production of ILT gG specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the ILT gG protein. Monoclonal antibodies aregenerated in mice utilizing this virus according to the PROCEDURE FORPURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials &Methods).

Example 11 S-SPV-016

S-SPV-016 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiouslaryngotracheitis virus glycoprotein I (ILT gI) were inserted into theSPV 617-48.1 ORF (a unique NotI restriction site has replaced a uniqueAccI restriction site). The lacZ gene is under the control of thesynthetic late promoter (LP1), and the ILT gI gene is under the controlof the synthetic late/early promoter (LP2EP2).

S-SPV-016 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 624-20.1C (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-016. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-016 was assayed for expression of ILT gI- andβ-galactosidase-specific antigens using the BLACK PLAQUE SCREEN FORFOREIGN GENE EXPRESSION IN RECOMBINANT SPV. Polyclonal chicken anti-ILTantibody was shown to react specifically with S-SPV-016 plaques and notwith S-SPV-017 negative control plaques. All S-SPV-016 observed plaquesreacted with the chicken antiserum indicating that the virus was stablyexpressing the ILT foreign gene. The assays described here were carriedout in ESK-4 cells, indicating that ESK-4 cells would be a suitablesubstrate for the production of SPV recombinant vaccines.

To confirm the expression of the ILT gI gene product, cells wereinfected with SPV-016 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal chickenanti-ILT antibody was used to detect expression of ILT specificproteins. The lysate from S-SPV-016 infected cells exhibits a range ofbands reactive to the anti-ILT antibody from 40 to 200 kd indicatingthat the ILT gI may be heavily modified.

This virus is used as an expression vector for expressing ILTglycoprotein I (gI). Such ILT gI is used as an antigen to identifyantibodies directed against the wild-type ILT virus as opposed toantibodies directed against gI deleted ILT viruses. This virus is alsoused as an antigen for the production of ILT gI specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the ILT gI protein.

Monoclonal antibodies are generated in mice utilizing this virusaccording to the PROCEDURE FOR PURIFICATION OF VIRAL GLYCOPROTEINS FORUSE AS DIAGNOSTICS (Materials & Methods).

Example 12 S-SPV-017

S-SPV-017 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiousbovine rhinotracheitis virus glycoprotein G (IBR gG) were inserted intothe SPV 617-48.1 ORF (a unique NotI restriction site has replaced aunique AccI restriction site). The lacZ gene is under the control of thesynthetic late promoter (LP1), and the IBR gG gene is under the controlof the synthetic late/early promoter (LP2EP2).

S-SPV-017 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 614-83.18 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-017. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-017 was assayed for expression of IBR-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Monoclonal antibodies and peptide antisera to IBR gG were shown to reactspecifically with S-SPV-017 plaques and not with S-SPV-016 negativecontrol plaques. All S-SPV-017 observed plaques reacted with theantiserum indicating that the virus was stably expressing the IBRforeign gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the IBR gG gene product, cells wereinfected with SPV-017 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Antisera to IBR gGwas used to detect expression of IBR specific proteins. The lysate fromS-SPV-017 infected cells exhibited a band at 43 kd which is the expectedsize of the IBR gC protein and additional bands of higher molecularweight which represent glycosylated forms of the protein which areabsent in deletion mutants for IBR gG.

This virus is used as an expression vector for expressing IBRglycoprotein G (gG). Such IBR gG is used as an antigen to identifyantibodies directed against the wild-type IBR virus as opposed toantibodies directed against gG deleted IBR viruses. This virus is alsoused as an antigen for the production of IBR gG specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the IBR gG protein. Monoclonal antibodies aregenerated in mice utilizing this virus according to the PROCEDURE FORPURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials &Methods).

Example 13 S-SPV-019

S-SPV-019 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacz) and the gene for infectiousbovine rhinotracheitis virus (IBRV) gE were inserted into the SPV617-48.1 ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the IBRV gE gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-019 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 708-78.9 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-019. This virus was assayed forβ-galactosidase expression, purity and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

This virus is used as an expression vector for expressing IBRglycoprotein E (gE). Such IBR gE is used as an antigen to identifyantibodies directed against the wild-type IBR virus as opposed toantibodies directed against gE deleted IBR viruses. This virus is alsoused as an antigen for the production of IBR gE specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the IBR gE protein. Monoclonal antibodies aregenerated in mice utilizing this virus according to the PROCEDURE FORPURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials &Methods).

Example 14 S-SPV-018

S-SPV-018 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacz) and the gene forpseudorabies virus glycoprotein E (PRV gE) are inserted into the SPV570-33.32 ORF (a unique PstI site has replaced the unique AccI site).The lacZ gene is under the control of the synthetic late promoter (LP1),and the PRV gE gene is under the control of the synthetic early/latepromoter (EP1LP2).

S-SPV-018 is derived from the S-SPV-001 (Kasza Strain). This isaccomplished utilizing the final homology vector and virus S-SPV-001 inthe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV.The transfection stock is screened by the SCREEN FOR RECOMBINANT SPVEXPRESSING β-galactosidase (BLUOGAL AND CPRG ASSAYS). Red plaquepurification of the recombinant virus is designated S-SPV-018. Thisvirus is assayed for β-galactosidase expression, purity, and insertstability by multiple passages monitored by the blue plaque assaydescribed in Materials and Methods.

After the initial three rounds of purification, all plaques observed areblue indicating that the virus is pure, stable, and expressing theforeign gene.

This virus is used as an expression vector for expressing PRVglycoprotein E (gE). Such PRV gE is used as an antigen to identifyantibodies directed against the wild-type PRV virus as opposed toantibodies directed against gE deleted PRV viruses. This virus is alsoused as an antigen for the production of PRV gE specific monoclonalantibodies. Such antibodies are useful in the development of diagnostictests specific for the PRV gE protein. Monoclonal antibodies aregenerated in mice utilizing this virus according to the PROCEDURE FORPURIFICATION OF VIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials &Methods).

Example 15 Homology Vector 520-90.15

The homology vector 520-90.15 is a plasmid useful for the insertion offoreign DNA into SPV. Plasmid 520-90.15 contains a unique NdeIrestriction site into which foreign DNA may be cloned. A plasmidcontaining such a foreign DNA insert has been used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV togenerate a SPV containing the foreign DNA. For this procedure to besuccessful, it is important that the insertion site be in a regionnon-essential to the replication of the SPV and that the site be flankedwith swinepox virus DNA appropriate for mediating homologousrecombination between virus and plasmid DNAs. The unique NdeIrestriction site in plasmid 520-90.15 is located within the codingregion of the SPV thymidine kinase gene (32). Therefore, thymidinekinase gene of swinepox virus was shown to be non-essential for DNAreplication and is an appropriate insertion site.

Example 16 S-PRV-010

S-SPV-010 is a swinepox virus that expresses a foreign gene. The E. coliβ-galactosidase (lacZ) gene is inserted into a unique NdeI restrictionsite within the thymidine kinase gene. The foreign gene (lacZ) is underthe control of the synthetic late promoter, LP1. Thus, swinepox virusthymidine kinase gene was shown to be non-essential for replication ofthe virus and is an appropriate insertion site.

A 1739 base pair HindIII-BamHI fragment subcloned from the HindIII Gfragment contains the swinepox virus thymidine kinase gene and isdesignated homology vector 520-90.15. The homology vector 520-90.15 wasdigested with Nde I, and AscI linkers were inserted at this unique sitewithin the thymidine kinase gene. The LP1 promoter-lac Z cassette withAscI linkers was ligated into the Asc I site within the thymidine kinasegene. The recombinant homology vector 561-36.26 was cotransfected withvirus S-SPV-001 by the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV and virus plaques expressing β-galactosidase wereselected by SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase(BLUOGAL AND CPRG ASSAY). The final result of blue and red plaquepurification was the recombinant virus designated S-SPV-010. This viruswas assayed for β-galactosidase expression, purity, and insert stabilityby multiple passages monitored by the blue plaque assay as described inMaterials and Methods. After the initial three rounds of purification,all plaques observed were blue indicating that the virus was pure,stable and expressing the foreign gene. The assays described here werecarried out in ESK-4 cells, indicating that ESK-4 cells would be asuitable substrate for the production of SPV recombinant vaccines.

Example 17

The development of vaccines utilizing the swinepox virus to expressantigens from various disease causing microorganisms can be engineered.

TRANSMISSIBLE GASTROENTERITIS VIRUS

The major neutralizing antigen of the transmissible gastroenteritisvirus (TGE), glycoprotein 195, for use in the swinepox virus vector hasbeen cloned. The clone of the neutralizing antigen is disclosed in U.S.Ser. No. 078,519, filed Jul. 27, 1987. It is contemplated that theprocedures that have been used to express PRV g50 (gD) in SPV and aredisclosed herein are applicable to TGE.

PORCINE PARVOVIRUS

The major capsid protein of the porcine (swine) parvovirus (PPV) wascloned for use in the swinepox virus vector. The clone of the capsidprotein is disclosed in U.S. Pat. No. 5,068,192 issued Nov. 26, 1991. Itis contemplated that the procedures that have been used to express PRVg50 (gD) in SPV and are disclosed herein are applicable to PPV.

SWINE ROTAVIRUS

The major neutralizing antigen of the swine rotavirus, glycoprotein 38,was cloned for use in the swinepox virus vector. The clone ofglycoprotein 38 is disclosed in U.S. Pat. No. 5,068,192 issued Nov. 26,1991. It is contemplated that the procedures that have been used toexpress PRV g50 (gD) in SPV and are disclosed herein are applicable toSRV.

HOG CHOLERA VIRUS

The major neutralizing antigen of the bovine viral diarrhea (BVD) viruswas cloned as disclosed in U.S. Ser. No. 225,032, filed Jul. 27, 1988.Since the BVD and hog cholera viruses are cross protective (31), the BVDvirus antigen has been targeted for use in the swinepox virus vector. Itis contemplated that the procedures that have been used to express PRVg50 (gD) in SPV and are disclosed herein are applicable to BVD virus.

SERPULINA HYODYSENTERIAE

A protective antigen of Serpulina hyodysenteriae (3), for use in theswinepox virus vector has been cloned. It is contemplated that theprocedures that have been used to express PRV g50 in SPV and aredisclosed herein are also applicable to Serpulina hyodysenteriae.

Antigens from the following microorganisms may also be utilized todevelop animal vaccines: swine influenza virus, foot and mouth diseasevirus, African swine fever virus, hog cholera virus, Mycoplasmahyopneumoniae, porcine reproductive and respiratory syndrome/swineinfertility and respiratory syndrome (PRRS/SIRS).

Antigens from the following microorganisms may also be utilized foranimal vaccines: 1) canine—herpesvirus, canine distemper, canineadenovirus type 1 (hepatitis), adenovirus type 2 (respiratory disease),parainfluenza, Leptospira canicola, icterohemorragia, parvovirus,coronavirus, Borrelia burgdorferi, canine herpesvirus, Bordetellabronchiseptica, Dirofilaria immitis (heartworm) and rabies virus. 2)Feline—Fiv gag and env, feline leukemia virus, feline immunodeficiencyvirus, feline herpesvirus, feline infectious peritonitis virus, canineherpesvirus, canine coronavirus, canine parvovirus, parasitic diseasesin animals (including Dirofilaria immitis in dogs and cats), equineinfectious anemia, Streptococcus equi, coccidia, emeria, chicken anemiavirus, Borrelia bergdorferi, bovine coronavirus, Pasteurellahaemolytica.

Example 17A

Vaccines containing recombinant swinepox virus expressing antigens fromhog cholera virus, swine influenza virus and (porcine reproducting andrespiratory syndrome) PRRS virus.

Recombinant swinepox virus expressing genes for neutralizing antigens tohog cholera virus, swine influenza virus and PRRS virus is useful toprevent disease in swine. The genes expressed in the recombinant SPVinclude, but are not limited to hog cholera virus gE1 and gE2 genes,swine influenza virus hemagglutinin, neuraminidase, matrix andnucleoprotein, and PRRS virus ORF7.

Example 18

Recombinant swinepox viruses express equine influenza virus typeA/Alaska 91, equine influenza virus type A/Prague 56, equine herpesvirustype 1 gB, or equine herpesvirus type 1 gD genes. S-SPV-033 andS-SPV-034 are useful as vaccines against equine influenza infection, andS-SPV-038 and S-SPV-039 are useful as a vaccine against equineherpesvirus infection which causes equine rhinotracheitis and equineabortion. These equine influenza and equine herpesvirus antigens are keyto raising a protective immune response in the animal. The recombinantviruses are useful alone or in combination as an effective vaccine. Theswinepox virus is useful for cloning other subtypes of equine influenzavirus (including equine influenza virus type A/Miami/63 and equineinfluenza virus type A/Kentucky/81) to protect against rapidly evolvingvariants in this disease. S-SPV-033, S-SPV-034, S-SPV-038, and S-SPV-039are also useful as an expression vector for expressing equine influenzaor equine herpesvirus antigens. Such equine influenza or equineherpesvirus antigens are useful to identify antibodies directed againstthe wild-type equine influenza virus or equine herpesvirus. The virusesare also useful to in producing antigens for the production ofmonospecific polyclonal or monoclonal antibodies. Such antibodies areuseful in the development of diagnostic tests specific for the viralproteins. Monoclonal or polyclonal antibodies are generated in miceutilizing these viruses according to the PROCEDURE FOR PURIFICATION OFVIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials and Methods).

Example 18A S-SPV-033

S-SPV-033 is a recombinant swinepox virus that expresses at least twoforeign genes. The gene for E. coli β-galactosidase (lacZ) and the genefor equine influenza virus type A/Alaska 91 neuraminidase were insertedinto the SPV 617-48.1 ORF (a unique NotI restriction site has replaced aunique AccI restriction site). The lacZ gene is under the control of thesynthetic late promoter (LP1), and the EIV AK/91 NA gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-SPV-033 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 732-18.4 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-033. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

Example 18B S-SPV-034

S-SPV-034 is a swinepqx virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for equineinfluenza virus type A/Prague 56 neuraminidase were inserted into theSPV 617-48.1 ORF (a unique NotI restriction site has replaced a uniqueAccI restriction site). The lacZ gene is under the control of thesynthetic late promoter (LP1), and the EIV PR/56 NA gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-SPV-034 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 723-59A9.22 (see Materialsand Methods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock wasscreened by the SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase(BLUOGAL AND CPRG ASSAYS). The final result of red plaque purificationwas the recombinant virus designated S-SPV-034. This virus was assayedfor β-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-034 was assayed for expression of EIV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Monospecific polyclonal antibodies to EIV PR/56 NA were shown to reactspecifically with S-SPV-034 plaques and not with S-SPV-001 negativecontrol plaques. All S-SPV-034 observed plaques reacted with theantiserum indicating that the virus was stably expressing the EIV PR/56NA gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

Example 18C S-SPV-038

S-SPV-038 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for equineherpesvirus type 1 glycoprotein B are inserted into the SPV 617-48.1 ORF(a unique NotI restriction site has replaced a unique AccI restrictionsite). The lacZ gene is under the control of the synthetic late promoter(LP1), and the EHV-1 gB gene is under the control of the syntheticlate/early promoter (LP2EP2).

S-SPV-038 is derived from S-SPV-001 (Kasza Strain). This is accomplishedutilizing the homology vector 744-34 (see Materials and Methods) andvirus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV. The transfection stock is screened by the SCREEN FORRECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL AND CPRG ASSAYS).The final result of red plaque purification is the recombinant virusdesignated S-SPV-038. This virus is assayed for β-galactosidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is pure, stable, and expressing the foreigngene.

Example 18D S-SPV-039

S-SPV-039 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for equineherpesvirus type 1 glycoprotein D are inserted into the SPV 617-48.1 ORF(a unique NotI restriction site has replaced a unique AccI restrictionsite). The lacZ gene is under the control of the synthetic late promoter(LP1), and the EHV-1 gD gene is under the control of the syntheticlate/early promoter (LP2EP2).

S-SPV-039 is derived from S-SPV-001 (Kasza Strain). This is accomplishedutilizing the homology vector 744-38 (see Materials and Methods) andvirus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV. The transfection stock is screened by the SCREEN FORRECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL AND CPRG ASSAYS).The final result of red plaque purification is the recombinant virusdesignated S-SPV-039. This virus is assayed for β-galactosidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is pure, stable, and expressing the foreigngene.

Example 19

Recombinant swinepox viruses express bovine respiratory syncytial virusattachment protein (BRSV G), BRSV Fusion protein (BRSV F), BRSVnucleocapsid protein (BRSV N), bovine viral diarrhea virus (BVDV) g48,BVDV g53, bovine parainfluenza virus type 3 (BPI-3) F, or BPI-3 HN.S-SPV-020, S-SPV-029, S-SPV-030, and S-SPV-032, S-SPV-028 are useful asvaccines against bovine disease. These BRSV, BVDV, and BPI-3 antigensare key to raising a protective immune response in the animal. Therecombinant viruses are useful alone or in combination as an effectivevaccine. The swinepox virus is useful for cloning other subtypes ofBRGV, BVDV, and BPI-3 to protect against rapidly evolving variants inthis disease. S-SPV-020, S-SPV-029, S-SPV-030, and S-SPV-032, S-SPV-028are also useful as an expression vector for expressing BRSV, BVDV, andBPI-3 antigens. Such BRSV, BVDV, and BPI-3 antigens are useful toidentify antibodies directed against the wild-type BRSV, BVDV, andBPI-3. The viruses are also useful as antigens for the production ofmonospecific polyclonal or monoclonal antibodies. Such antibodies areuseful in the development of diagnostic tests specific for the viralproteins. Monoclonal or polyclonal antibodies are generated in miceutilizing these viruses according to the PROCEDURE FOR PURIFICATION OFVIRAL GLYCOPROTEINS FOR USE AS DIAGNOSTICS (Materials and Methods).

Example 19A S-SPV-020

S-SPV-020 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacz) and the gene for bovinerespiratory syncytial virus (BRSV) G were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the BRSV G gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-020 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 727-20.5 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-020. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-020 was assayed for expression of BRSV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Bovine anti-BRSV FITC (Accurate Chemicals) was shown to reactspecifically with S-SPV-020 plaques and not with S-SPV-003 negativecontrol plaques. All S-SPV-020 observed plaques reacted with theantiserum indicating that the virus was stably expressing the BRSVforeign gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the BRSV G gene product, cells wereinfected with S-SPV-020 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Bovine anti-BRSV FITC(Accurate Chemicals) was used to detect expression of BRSV specificproteins. The lysate from S-SPV-020 infected cells exhibited a band at36 kd which is the expected size of the non-glycosylated form of BRSV Gprotein and bands at 43 to 45 kd and 80 to 90 kd which are the expectedsize of glycosylated forms of the BRSV G protein.

Example 19B S-SPV-029

S-SPV-029 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovinerespiratory syncytial virus (BRSV) F were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the BRSV F gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-029 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 727-20.10 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-029. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods.

After the initial three rounds of purification, all plaques observedwere blue indicating that the virus was pure, stable, and expressing theforeign gene.

S-SPV-029 was assayed for expression of BRSV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Bovine anti-BRSV FITC (Accurate Chemicals) was shown to reactspecifically with S-SPV-029 plaques and not with S-SPV-003 negativecontrol plaques. All S-SPV-029 observed plaques reacted with theantiserum indicating that the virus was stably expressing the BRSVforeign gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

Example 19C S-SPV-030

S-SPV-030 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovinerespiratory syncytial virus (BRSV) N were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the BRSV N gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-030 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 713-55.37 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-030. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-030 was assayed for expression of BRSV-specific antigens using theBLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Bovine anti-BRSV FITC (Accurate Chemicals) was shown to reactspecifically with S-SPV-030 plaques and not with S-SPV-003 negativecontrol plaques. All S-SPV-030 observed plaques reacted with theantiserum indicating that the virus was stably expressing the BRSVforeign gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the BRSV N gene product, cells wereinfected with SPV-030 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Bovine anti-BRSV FITC(Accurate Chemicals) was used to detect expression of BRSV specificproteins. The lysate from S-SPV-030 infected cells exhibited a band at43 kd which is the expected size of the BRSV N protein.

Example 19D S-SPV-028

S-SPV-028 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovineparainfluenza virus type 3 (BPI-3) F were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the BPI-3 F gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-028 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 713-55.10 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-028. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-028 was assayed for expression of BPI-3-specific antigens usingthe BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Bovine anti-BPI-3 FITC (Accurate Chemicals) was shown to reactspecifically with S-SPV-028 plaques and not with S-SPV-003 negativecontrol plaques. All S-SPV-028 observed plaques reacted with theantiserum indicating that the virus was stably expressing the BPI-3foreign gene. The assays described here were carried out in ESK-4 cells,indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the BPI-3 F gene product, cells wereinfected with SPV-028 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Bovine anti-BPI-3FITC (Accurate Chemicals) was used to detect expression of BPI-3specific proteins. The lysate from S-SPV-028 infected cells exhibitedbands at 43, and 70 kd which is the expected size of the BPI-3 Fprotein.

Example 19E S-SPV-032

S-SPV-032 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovineviral diarrhea virus (BVDV) g48 were inserted into the SPV 617-48.1 ORF(a unique NotI restriction site has replaced a unique AccI restrictionsite). The lacZ gene is under the control of the synthetic late promoter(LP1), and the BVDV g48 gene is under the control of the syntheticlate/early promoter (LP2EP2).

S-SPV-032 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 727-78.1 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-032. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods.

After the initial three rounds of purification, all plaques observedwere blue indicating that the virus was pure, stable, and expressing theforeign gene.

Example 19F S-SPV-040

S-SPV-040 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovineviral diarrhea virus (BVDV) g53 were inserted into the SPV 617-48.1 ORF(a unique NotI restriction site has replaced a unique AccI restrictionsite). The lacz gene is under the control of the synthetic late promoter(LP1), and the BVDV g53 gene is under the control of the syntheticlate/early promoter (LP2EP2).

S-SPV-040 is derived from S-SPV-001 (Kasza Strain). This is accomplishedutilizing the homology vector 738-96 (see Materials and Methods) andvirus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT SPV. The transfection stock is screened by the SCREEN FORRECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL AND CPRG ASSAYS).The final result of red plaque purification is the recombinant virusdesignated S-SPV-040. This virus is assayed for β-galactosidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is pure, stable, and expressing the foreigngene.

Example 19G Shipping Fever Vaccine

Shipping fever or bovine respiratory disease (BRD) complex is manifestedas the result of a combination of infectious diseases of cattle andadditional stress related factors (52). Respiratory virus infectionsaugmented by pathophysiological effects of stress, alter thesusceptibility of cattle to Pasteurella organisms by a number ofmechanisms. Control of the viral infections that initiate BRD isessential to preventing the disease syndrome (53).

The major infectious disease pathogens that contribute to BRD includebut are not limited to infectious bovine rhinotracheitis virus (IBRV),parainfluenza virus type 3 (PI-3), bovine respiratory syncytial virus(BRSV), and Pasteurella haemolytica (53). Recombinant swinepox virusexpressing protective antigens to organisms causing BRD is useful as avaccine. S-SPV-020, S-SPV-029, S-SPV-030, S-SPV-032, and S-SPV-028 areuseful components of such a vaccine.

Example 20

Recombinant swinepox viruses S-SPV-031 and S-SPV-035 are useful as avaccine against human disease. S-SPV-031 expresses the core antigen ofhepatitis B virus. S-SPV-031 is useful against hepatitis B infection inhumans. S-SPV-035 expresses the cytokine, interleukin-2, and is usefulas an immune modulator to enhance an immune response in humans. WhenS-SPV-031 and S-SPV-035 are combined, a superior vaccine againsthepatitis B is produced.

Example 20A S-SPV-031

S-SPV-031 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for Hepatitis BCore antigen were inserted into the SPV 617-48.1 ORF (a unique NotIrestriction site has replaced a unique AccI restriction site). The laczgene is under the control of the synthetic late promoter (LP1), and theHepatitis B Core antigen gene is under the control of the syntheticearly/late promoter (EP1LP2).

S-SPV-031 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 727-67.18 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-031. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-031 was assayed for expression of Hepatitis B Coreantigen-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENEEXPRESSION IN RECOMBINANT SPV. Rabbit antisera to Hepatitis B Coreantigen was shown to react specifically with S-SPV-031 plaques and notwith S-SPV-001 negative control plaques. All S-SPV-031 observed plaquesreacted with the antiserum indicating that the virus was stablyexpressing the Hepatitis B Core antigen gene. The assays described herewere carried out in ESK-4 cells, indicating that ESK-4 cells would be asuitable substrate for the production of SPV recombinant vaccines.

To confirm the expression of the Hepatitis B Core antigen gene product,cells were infected with SPV-031 and samples of infected cell lysateswere subjected to SDS-polyacrylamide gel electrophoresis. The gel wasblotted and analyzed using the WESTERN BLOTTING PROCEDURE. Rabbitantisera to Hepatitis B Core antigen was used to detect expression ofHepatitis B specific proteins. The lysate from S-SPV-031 infected cellsexhibited a band at 21 kd which is the expected size of the Hepatitis BCore antigen.

Example 20B S-SPV-035

S-SPV-035 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for human IL-2were inserted into the SPV 617-48.1 ORF (a unique NotI restriction sitehas replaced a unique AccI restriction site). The lacZ gene is under thecontrol of the synthetic late promoter (LP1), and the human IL-2 gene isunder the control of the synthetic late/early promoter (LP2EP2).

S-SPV-035 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 741-84.14 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-035. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

Example 21 Human Vaccines Using Recombinant Swinepox Virus as a Vector

Recombinant swinepox virus is useful as a vaccine against humandiseases. For example, human influenza virus is a rapidly evolving viruswhose neutralizing viral epitopes rapidly change. A useful recombinantswinepox vaccine is one in which the influenza virus neutralizingepitopes are quickly adapted by recombinant DNA techniques to protectagainst new strains of influenza virus. Human influenza virushemagglutinin (HN) and neuraminidase (NA) genes are cloned into theswinepox virus as described in CLONING OF EQUINE INFLUENZA VIRUSHEMAGGLUTININ AND NEURAMINIDASE GENES (See Materials and Methods andExample 17).

Recombinant swinepox virus is useful as a vaccine against other humandiseases when foreign antigens from the following diseases or diseaseorganisms are expressed in the swinepox virus vector: hepatitis B virussurface and core antigens, hepatitis C virus, human immunodeficiencyvirus, human herpesviruses, herpes simplex virus-1, herpes simplexvirus-2, human cytomegalovirus, Epstein-Barr virus, Varicella-Zostervirus, human herpesvirus-6, human herpesvirus-7, human influenza,measles virus, hantaan virus, pneumonia virus, rhinovirs, poliovirus,human respiratory syncytial virus, retrovirus, human T-cell leukemiavirus, rabies virus, mumps virus, malaria (Plasmodium falciparum),Bordetelia pertussis, Diptheria, Rickettsia prowazekii, Borreliabergdorferi, Tetanus toxoid, malignant tumor antigens.

Furthermore, S-SPV-035 (Example 20), when combined with swinepox virusinterleukin-2 is useful in enhancing immune response in humans.Additional cytokines, including but not limited to, interleukin-2,interleukin-6, interleukin-12, interferons, granulocyte-macrophagecolony stimulating factors, interleukin receptors from human and otheranimals when vectored into a non-essential site in the swinepox viralgenome, and subsequently expressed, have immune stimulating effects.

Recombinant swinepox virus express foreign genes in a human cell line.S-SPV-003 (EP1LP2 promoter expressing the lacZ gene) expressed the lacZgene in THP human monocyte cell lines by measuring β-galactosidaseactivity. Cytopathic effect of swinepox virus was observed on the THPhuman monocyte cells, indicating that recombinant swinepox virus canexpress foreign genes in a human cell line, but will not productivelyinfect or replicated in the human cell line. Swinepox virus wasdemonstrated to replicate well in ESK-4 cells (embryonic swine kidney)indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

Example 22

Avian vaccines using recombinant swinepox virus as a vector.

Example 22A S-SPV-026

S-SPV-026 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiousbursal disease virus (IBDV) polyprotein were inserted into the SPV617-48.1 ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the IBDV polyprotein gene is under the controlof the synthetic early/late promoter (EP1LP2).

S-SPV-026 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 689-50.4 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-026. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indication that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-026 was assayed for expression of IBDV polyprotein-specificantigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION INRECOMBINANT SPV. Rat antisera to IBDV polyprotein were shown to reactspecifically with S-SPV-026 plaques and not with S-SPV-001 negativecontrol plaques. All S-SPV-026 observed plaques reacted with theantiserum indicating that the virus was stably expressing the IBDVpolyprotein gene. The assays described here were carried out in ESK-4cells, indicating that ESK-4 cells would be a suitable substrate for theproduction of SPV recombinant vaccines.

To confirm the expression of the IBDV polyprotein gene product, cellswere infected with SPV-026 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Rat antisera to IBDVproteins VP2, VP1, and VP4 and monoclonal antibody R63 to IBDV VP2 wereused to detect expression of IBDV proteins. The lysate from S-SPV-026infected cells exhibited bands at 32 to 40 kd which is the expected sizeof the IBDV proteins.

Example 22B S-SPV-027

S-SPV-027 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiousbursal disease virus (IBDV) VP2 (40 kd) were inserted into the SPV617-48.1 ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the IBDV VP2 gene is under the control of thesynthetic early/late promoter (EP1LP2).

S-SPV-027 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 689-50.7 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-027. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-027 was assayed for expression of IBDV VP2-specific antigens usingthe BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT SPV.Rat antisera to IBDV protein was shown to react specifically withS-SPV-027 plaques and not with S-SPV-001 negative control plaques. AllS-SPV-027 observed plaques reacted with the antiserum indicating thatthe virus was stably expressing the IBDV VP2 gene. The assays describedhere were carried out in ESK-4 cells, indicating that ESK-4 cells wouldbe a suitable substrate for the production of SPV recombinant vaccines.

To confirm the expression of the IBDV VP2 gene product, cells wereinfected with S-SPV-027 and samples of infected cell lysates weresubjected to SDS-polyacrylamide gel electrophoresis. The gel was blottedand analyzed using the WESTERN BLOTTING PROCEDURE. Rat antisera to IBDVprotein and monoclonal antibody R63 to IBDV VP2 were used to detectexpression of IBDV VP2 protein. The lysate from S-SPV-027 infected cellsexhibited a band at 40 kd which is the expected size of the IBDV VP2protein.

S-SPV-026 and S-SPV-027 are useful as vaccines against infectious bursaldisease in chickens and also as expression vectors for IBDV proteins.Recombinant swinepox virus is useful as a vaccine against other aviandisease when foreign antigens from the following diseases or diseaseorganisms are expressed in the swinepox virus vector: Marek's diseasevirus, infectious laryngotracheitis virus, Newcastle disease virus,infectious bronchitis virus, and chicken anemia virus, Chick anemiavirus, Avian encephalomyelitis virus, Avian reovirus, Avianparamyxoviruses, Avian influenza virus, Avian adenovirus, Fowl poxvirus, Avian coronavirus, Avian rotavirus, Salmonella spp E coli,Pasteurella spp, Haemophilus spp, Chlamydia spp, Mycoplasma spp,Campylobacter spp, Bordetella spp, Poultry nematodes, cestodes,trematodes, Poultry mites/lice, Poultry protozoa (Eimeria spp,Histomonas spp, Trichomonas spp).

Example 23 SPV-036

S-SPV-036 is a swinepox virus that expresses at one foreign gene. Thegene for E. coli β-galactosidase (lacZ) was inserted into the SPV617-48.1 ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the humancytomegalovirus immediate early (HCMV IE) promoter.

S-SPV-036 was derived from S-SPV-001 (Kasza Strain) This wasaccomplished utilizing the homology vector 741-80.3 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-036. This virus is assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved are blue indicating that the virus is pure, stable, andexpressing the foreign gene.

The expression of lacz from the HCMV IE promoter provides a strongpromoter for expression of foreign genes in swinepox. S-SPV-036 is anovel and unexpected demonstration of a herpesvirus promoter drivingexpression of a foreign gene in a poxvirus. S-SPV-036 is useful informulating human vaccines, and recombinant swinepox virus is useful forthe expression of neutralizing antigens from human pathogens.Recombinant swinepox virus expressed foreign genes in a human cell lineas demonstrated by S-SPV-003 (EP1LP2) promoter expressing the lacZ gene)expressed β-galactosidase in THP human monocyte cell lines. Cytopathiceffects of swinepox virus on the THP human monocyte cells were notobserved, indicating that recombinant swinepox virus can express foreigngenes in a human cell line, but will not productively infect orreplicated in the human cell line

Example 24 Homology Vector 738-94.4

Homology Vector 738-94.4 is a swinepox virus vector that expresses oneforeign gene. The gene for E. coli β-galactosidase (lacZ) was insertedinto the the O1L open reading frame (SEQ ID NO: 115). The lacZ gene isunder the control of the O1L promoter. The homology vector 738-94.4contains a deletion of SPV DNA from nucleotides 1679 to 2452 (SEQ ID NO:189; FIG. 17) which deletes part of the O1L ORF.

The upstream SPV sequences were synthesized by polymerase chain reactionusing DNA primers 5′-GAAGCATGCCCGTTCTTATCAATAGTTTAGTCGAAAATA-3′, (SEQ IDNO: 185) and 5′-CATAAGATCTGGCATTGTGTTATTATACTAACAAAAATAAG-3′ (SEQ ID NO:186) to produce an 855 base pair fragment with BglII and SphI ends. TheO1L promoter is present on this fragment. The downstream SPV sequenceswere synthesized by polymerase chain reaction using DNA primers5′-CCGTAGTCGACAAAGATCGACTTATTAATATGTATGGGATT-3′ (SEQ ID NO: 187) and5′-GCCTGAAGCTTCTAGTACAGTATTTACGACTTTTGAAAT-3′ (SEQ ID NO: 188) toproduce an 1113 base pair fragment with SalI and HindIII ends. Arecombinant swinepox virus was derived utilizing homology vector738-94.4 and S-SPV-001 (Kasza strain) in the HOMOLOGOUS RECOMBINATIONPROCEDURE FOR GENERATING RECOMBINANT SPV. The transfection stock wasscreened by the SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase(BLUOGAL AND CPRG ASSAYS). The final result of red plaque purificationis the recombinant virus. This virus is assayed for β-galactosidaseexpression, purity, and insert stability by multiple passages monitoredby the blue plaque assay as described in Materials and Methods. Afterthe initial three rounds of purification, all plaques observed are blueindicating that the virus is pure, stable, and expressing the foreigngene. Recombinant swinepox viruses derived from homology vector 738-94.4are utilized as an expression vector to express foreign antigens and asa vaccine to raise a protective immune response in animals to foreigngenes expressed by the recombinant swinepox virus. Other promoters inaddition to the O1L promoter are inserted into the deleted regionincluding LP1, EP1LP2, LP2EP2, HCMV immediate early, and one or moreforeign genes are expressed from these promoters.

Example 24B

Homology Vector 752-22.1 is a swinepox virus vector that is utilized toexpress two foreign genes. The gene for E. coli β-galactosidase (lacZ)was inserted into the the O1L open reading frame (SEQ ID NO: 115). ThelacZ gene is under the control of the O1L promoter. A second foreigngene is expressed from the LP2EP2 promoter inserted into an EcoRI orBamHI site following the LP2EP2 promoter sequence. The homology vector752-22.1 contains a deletion of SPV DNA from nucleotides 1679 to 2452(SEQ ID NO: 189; FIG. 17) which deletes part of the 01L ORF. Thehomology vector 752-22.1 was derived from homology vector 738-94.4 byinsertion of the LP2EP2 promoter fragment (see Materials and Methods).The homology vector 752-22.1 is further improved by placing the lacZgene under the control of the synthetic LP1 promoter. The LP1 promoterresults in higher levels of lacZ expression compared to the SPV O1Lpromoter

Example 25 S-SPV-041

S-SPV-041 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for equineherpesvirus type 1 glycoprotein B (gB) were inserted into the 738-94.4ORF (a 773 base pair deletion of the SPV O1L ORF; Deletion ofnucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ gene is under thecontrol of the swinepox O1L promoter, and the EHV-1 gB gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-SPV-041 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 752-29.33 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-041. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-041 is useful as a vaccine in horses against EHV-1 infection andis useful for expression of EHV-1 glycoprotein B.

S-SPV-045

S-SPV-045 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for infectiousbovine rhinotracheitis virus glycoprotein E (gE) were inserted into the738-94.4 ORF (a 773 base pair deletion of the SPV O1L ORF; Deletion ofnucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ gene is under thecontrol of the swinepox O1L promoter, and the IBRV gE gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-SPV-045 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 746-94.1 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-045. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-045 is useful for expression of IBRV glycoprotein E.

S-SPV-049

S-SPV-049 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for bovineviral diarrhea virus glycoprotein 48 (gp48) were inserted into the738-94.4 ORF (a 773 base pair deletion of the SPV O1L ORF; Deletion ofnucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ gene is under thecontrol of the swinepox O1L promoter, and the BVDV gp48 gene is underthe control of the synthetic late/early promoter (LP2EP2).

S-SPV-049 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 771-55.11 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-049. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-049 is useful as a vaccine in cattle against BVDV infection and isuseful for expression of BVDV glycoprotein 48.

S-SPV-050

S-SPV-050 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for the bovineviral diarrhea virus glycoprotein 53 (gp53) were inserted into the738-94.4 ORF (a 773 base pair deletion of the SPV O1L ORF; Deletion ofnucleotides 1679 to 2452, SEQ ID NO: 189). The lacZ gene is under thecontrol of the swinepox O1L promoter, and the IBRV gE gene is under thecontrol of the synthetic late/early promoter (LP2EP2).

S-SPV-050 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 767-67.3 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-050. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-050 is useful as a vaccine in cattle against BVDV infection and isuseful for expression of BVDV glycoprotein 53.

Example 26

Recombinant swinepox virus, S-SPV-042 or S-SPV-043, expressing chickeninterferon (cIFN) or chicken myelomonocytic growth factor (cMGF),respectively, are useful to enhance the immune response when added tovaccines against diseases of poultry. Chicken myelomonocytic growthfactor (cMGF) is homologous to mammalian interleukin-6 protein, andchicken interferon (cIFN) is homologous to mammalian interferon. Whenused in combination with vaccines against specific avian diseases,S-SPV-042 and S-SPV-043 provide enhanced mucosal, humoral, or cellmediated immunity against avian disease-causing viruses including, butnot limited to, Marek's disease virus, Newcastle disease virus,infectious laryngotracheitis virus, infectious bronchitis virus,infectious bursal disease virus.

Example 26A S-SPV-042

S-SPV-042 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for chickeninterferon (cIFN) were inserted into the SPV 617-48.1 ORF (a unique NotIrestriction site has replaced a unique AccI restriction site). The lacZgene is under the control of the synthetic late promoter (LP1), and thecIFN gene is under the control of the synthetic late/early promoter(LP2EP2).

S-SPV-042 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 751-07.Al (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-042. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-042 has interferon activity in cell culture. Addition of S-SPV-042conditioned media to chicken embryo fibroblast (CEF) cell cultureinhibits infection of the CEF cells by vesicular stomatitis virus or byherpesvirus of turkeys. S-SPV-042 is useful to enhance the immuneresponse when added to vaccines against diseases of poultry.

Example 26B S-SPV-043

S-SPV-043 is a swinepox virus that expresses at least two foreign genes.The gene for E. coli β-galactosidase (lacZ) and the gene for chickenmyelomonocytic growth factor (cMGF) were inserted into the SPV 617-48.1ORF (a unique NotI restriction site has replaced a unique AccIrestriction site). The lacZ gene is under the control of the syntheticlate promoter (LP1), and the cMGF gene is under the control of thesynthetic late/early promoter (LP2EP2).

S-SPV-043 was derived from S-SPV-001 (Kasza Strain). This wasaccomplished utilizing the homology vector 751-56.A1 (see Materials andMethods) and virus S-SPV-001 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT SPV. The transfection stock was screened bythe SCREEN FOR RECOMBINANT SPV EXPRESSING β-galactosidase (BLUOGAL ANDCPRG ASSAYS). The final result of red plaque purification was therecombinant virus designated S-SPV-043. This virus was assayed forβ-galactosidase expression, purity, and insert stability by multiplepassages monitored by the blue plaque assay as described in Materialsand Methods. After the initial three rounds of purification, all plaquesobserved were blue indicating that the virus was pure, stable, andexpressing the foreign gene.

S-SPV-043 is useful to enhance the immune response when added tovaccines against diseases of poultry.

Example 27 Insertion Into a Non-essential Site in the 2.0 kb HindIII toBqlII Region of the Swinepox Virus HindIII M Fragment

A 2.0 kb HindIII to BglII region of the swinepox virus HindIII Mfragment is useful for the insertion of foreign DNA into SPV. Theforeign DNA is inserted into a unique BglII restriction site in theregion (FIG. 17; Nucleotide 540 of SEQ ID NOs: 195). A plasmidcontaining a foreign DNA insert is used according to the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT SPV to generate anSPV containing the foreign DNA. For this procedure to be successful, itis important that the insertion site be in a region non-essential to thereplication of the SPV and that the site be flanked with swinepox virusDNA appropriate for mediating homologous recombination between virus andplasmid DNAs. The unique BglII restriction site in the 2.0 kb HindIII toBglII region of the swinepox virus HindIII M fragment is located withinthe coding region of the SPV I4L open reading frame. The I4L ORF hassequence similarity to the vaccinia virus and smallpox virusribonucleotide reductase (large subunit) gene (56-58). Theribonucleotide reductase (large subunit) gene is non-essential for DNAreplication of vaccinia virus and is an appropriate insertion site inswinepox virus.

REFERENCES

1. C. Bertholet, et al., EMBO Journal 5, 1951-1957 (1986).

2. R. A. Bhat, et al., Nucleic Acids Research 17, 1159-1176 (1989).

3. D. A. Boyden, et al., Infection and Immunity 57, 3808-3815 (1989).

4. D. B. Boyle and B. E. H. Coupar, Virus Research 10, 343-356 (1988).

5. R. M. Buller, et al., Nature 317, 813-815 (1985).

6. K. J. Cremer, et al., Science 228, 737-739 (1985).

7. A. J. Davidson and B. Moss, J. Mol. Biol. 210, 749-769 (1989).

8. A. J. Davidson and B. Moss, J. Mol. Biol. 210, 771-784 (1989).

9. P. L. Earl, et al., Journal of Virology 64, 2448-2451 (1990).

10. J. J. Esposito, et al., Virology 165, 313 (1988).

11. F. A. Ferrari, et al., J. of Bacteriology 161, 556-562 (1985).

12. C. Flexner, et al., Vaccine 8, 17-21 (1990).

13. S. J. Goebel, et al., Virology 179, 247-266 (1990).

14. U. Gubler and B. J. Hoffman, Gene 25, 263-269 (1983).

15. M. A. Innis, et al., PCR Protocols A Guide to Methods andApplications, 84-91, Academic Press, Inc., San Diego (1990).

16. S. Joshi, et al., Journal of Virology. 65, 5524-5530 (1991).

17. L. Kasza, et al., Am. J. Vet. Res. 21, 269-273 (1960).

18. L. Kasza, Diseases of Swine, 254-260, Ed. A. D. Leman, et al., TheIowa State University Press, Ames, Iowa (1981).

19. B. G. Klupp and T. C. Mettenleiter, Virology 182, 732-741 (1991).

20. U. K. Laemnli, Nature 227, 680-685 (1970).

21. B. Lominiczi, et al., Journal of Virology 49, 970-979 (1984).

22. T. Maniatis, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1982).

23. R. F. Massung, and R. W. Moyer, Virology 180, 347-354 (1991).

24. R. F. Massung, and R. W. Moyer, Virology 180, 355-364 (1991).

25. B. Moss, Science 252, 1662-1667 (1991).

26. E. A. Petrovskis, et al., Journal of Virology 59, 216-223 (1986).

27. A. K. Robbins et al., Journal of Virology 58, 339-347 (1986).

28. A. K. Robbins et al., Journal of Virology 61, 2691-2701 (1987).

29. A. C. R. Samson, Journal of Virology 67, 1199-1203 (1986).

30. J. Sambrook, et al., Molecular Cloning A Laboratory Manual SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

31. Sheffy, et al., Proceedings 65th Annual Meeting of the United StatesLivestock Association 65, 347-353 (1961).

32. W. M. Schnitzlein and D. N. Tripathy, Virology 181, 727-732, (1991).

33. J. Taylor, et al., Vaccine 9, 190-193, (1991).

34. M. Wachsman, et al., Journal of General Virology 70, 2513-2520(1989).

35. M. W. Wathen, et al., Journal of Virology 51, 57-62 (1984).

36. M. Weerasinge, Journal of Virology 65, 5531-5534 (1991).

37. T. Ben-Porat, et al., Journal of Virology, volume 154, 325-334(1986).

38. F. Zuckerman, et al., Vaccination and Control of Adjesky's Disease,J. T. Van Oirchot (ed.). Kluwer Academic Publishers, London, pp. 107-117(1989).

39. Paolette, et al., Journal of Virology, volume 66, pp. 3424-3434(June, 1992).

40. M. W. Mellencamp, et al., Journal of Clinical Microbiology, volume27, pp. 2208-2213 (1989).

41. L. A. Herzenberg, et al., Selected Methods in Cellular Immunology,Freeman Publ. Co., San Francisco, 351-372 (1980).

42. Katz et al., Journal of Virology 64, 1808-1811 (1990).

43. Taniguchi, T., et al., Biochem. Biophys. Res. Commun. 115 1040-1047(1983).

44. Cochran, M. D. and Macdonald, R. D., WO 93/02104, published Feb. 4,1993.

45. Galibert, F., et al., Nature 281, 646-650 (1979).

46. Thomsen, D. R., et al., Proc. Natl. Acad. Sci. USA 81, 659-663(1984).

47. Catalog Number 267402, Beckman Instruments, Inc., Fullerton Calif.

48. Whalley, J. M., et al., Journal of General Virology 57 307-323(1981).

49. Collett, M. S., et al., Virology 165 200-208 (1988).

50. Schodel, F. et al., Journal of Virology 66, 106-114 (1992).

51. Cochran, M. D., WO 93/25665, published Dec. 23, 1993.

52. C. A. Hjerpe, The bovine Respiratory Disease Complex. Ed. by J. L.Howard, Philadelphia, W. B. Saunders Co., 670-680 (1986).

53. F. Fenner, et al., Veterinary Virology. Academic Press, Inc.,Orlando Fla., 183-202 (1987).

54. A. Leutz, et al., EMBO Journal 8: 175-182 (1989).

55. M. J. Sekellick, et al., Journal of Interferon Research 14: 71-79(1994).

56. S. J. Child, et al., Virology 174: 625-629 (1990).

57. G. P. Johnson, et al. Virology 196: 381-401 (1993).

58. R. F Massung, et al. Virology 201: 215-240 (1994).

220 599 base pairs nucleic acid double linear DNA (genomic) NO NOSwinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS 202..597 /partial/codon_start= 202 /function= “Potential eukaryotic transcriptionalregulatory protein” /standard_name= “515-85.1 ORF” 1 AATGTATCCAGAGTTGTTGA ATGCCTTATC GTACCTAATA TTAATATAGA GTTATTAACT 60 GAATAAGTATATATAAATGA TTGTTTTTAT AATGTTTGTT ATCGCATTTA GTTTTGCTGT 120 ATGGTTATCATATACATTTT TAAGGCCGTA TATGATAAAT GAAAATATAT AAGCACTTAT 180 TTTTGTTAGTATAATAACAC A ATG CCG TCG TAT ATG TAT CCG AAG AAC GCA 231 Met Pro Ser TyrMet Tyr Pro Lys Asn Ala 1 5 10 AGA AAA GTA ATT TCA AAG ATT ATA TCA TTACAA CTT GAT ATT AAA AAA 279 Arg Lys Val Ile Ser Lys Ile Ile Ser Leu GlnLeu Asp Ile Lys Lys 15 20 25 CTT CCT AAA AAA TAT ATA AAT ACC ATG TTA GAATTT GGT CTA CAT GGA 327 Leu Pro Lys Lys Tyr Ile Asn Thr Met Leu Glu PheGly Leu His Gly 30 35 40 AAT CTA CCA GCT TGT ATG TAT AAA GAT GCC GTA TCATAT GAT ATA AAT 375 Asn Leu Pro Ala Cys Met Tyr Lys Asp Ala Val Ser TyrAsp Ile Asn 45 50 55 AAT ATA AGA TTT TTA CCT TAT AAT TGT GTT ATG GTT AAAGAT TTA ATA 423 Asn Ile Arg Phe Leu Pro Tyr Asn Cys Val Met Val Lys AspLeu Ile 60 65 70 AAT GTT ATA AAA TCA TCA TCT GTA ATA GAT ACT AGA TTA CATCAA TCT 471 Asn Val Ile Lys Ser Ser Ser Val Ile Asp Thr Arg Leu His GlnSer 75 80 85 90 GTA TTA AAA CAT CGT AGA GCG TTA ATA GAT TAC GGC GAT CAAGAC ATT 519 Val Leu Lys His Arg Arg Ala Leu Ile Asp Tyr Gly Asp Gln AspIle 95 100 105 ATC ACT TTA ATG ATC ATT AAT AAG TTA CTA TCG ATA GAT GATATA TCC 567 Ile Thr Leu Met Ile Ile Asn Lys Leu Leu Ser Ile Asp Asp IleSer 110 115 120 TAT ATA TTA GAT AAA AAA ATA ATT CAT GTA AC 599 Tyr IleLeu Asp Lys Lys Ile Ile His Val 125 130 132 amino acids amino acidlinear protein not provided 2 Met Pro Ser Tyr Met Tyr Pro Lys Asn AlaArg Lys Val Ile Ser Lys 1 5 10 15 Ile Ile Ser Leu Gln Leu Asp Ile LysLys Leu Pro Lys Lys Tyr Ile 20 25 30 Asn Thr Met Leu Glu Phe Gly Leu HisGly Asn Leu Pro Ala Cys Met 35 40 45 Tyr Lys Asp Ala Val Ser Tyr Asp IleAsn Asn Ile Arg Phe Leu Pro 50 55 60 Tyr Asn Cys Val Met Val Lys Asp LeuIle Asn Val Ile Lys Ser Ser 65 70 75 80 Ser Val Ile Asp Thr Arg Leu HisGln Ser Val Leu Lys His Arg Arg 85 90 95 Ala Leu Ile Asp Tyr Gly Asp GlnAsp Ile Ile Thr Leu Met Ile Ile 100 105 110 Asn Lys Leu Leu Ser Ile AspAsp Ile Ser Tyr Ile Leu Asp Lys Lys 115 120 125 Ile Ile His Val 130 899base pairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G CDS 3..662 /partial /codon_start= 3/function= “Potential eukaryotic transcriptional regulatory protein”/standard_name= “515-85.1 ORF” 3 GA GAT ATT AAA TCA TGT AAA TGC TCG ATATGT TCC GAC TCT ATA ACA 47 Asp Ile Lys Ser Cys Lys Cys Ser Ile Cys SerAsp Ser Ile Thr 1 5 10 15 CAT CAT ATA TAT GAA ACA ACA TCA TGT ATA AATTAT AAA TCT ACC GAT 95 His His Ile Tyr Glu Thr Thr Ser Cys Ile Asn TyrLys Ser Thr Asp 20 25 30 AAT GAT CTT ATG ATA GTA TTG TTC AAT CTA ACT AGATAT TTA ATG CAT 143 Asn Asp Leu Met Ile Val Leu Phe Asn Leu Thr Arg TyrLeu Met His 35 40 45 GGG ATG ATA CAT CCT AAT CTT ATA AGC GTA AAA GGA TGGGGT CCC CTT 191 Gly Met Ile His Pro Asn Leu Ile Ser Val Lys Gly Trp GlyPro Leu 50 55 60 ATT GGA TTA TTA ACG GGT GAT ATA GGT ATT AAT TTA AAA CTATAT TCC 239 Ile Gly Leu Leu Thr Gly Asp Ile Gly Ile Asn Leu Lys Leu TyrSer 65 70 75 ACC ATG AAT ATA AAT GGG CTA CGG TAT GGA GAT ATT ACG TTA TCTTCA 287 Thr Met Asn Ile Asn Gly Leu Arg Tyr Gly Asp Ile Thr Leu Ser Ser80 85 90 95 TAC GAT ATG AGT AAT AAA TTA GTC TCT ATT ATT AAT ACA CCC ATATAT 335 Tyr Asp Met Ser Asn Lys Leu Val Ser Ile Ile Asn Thr Pro Ile Tyr100 105 110 GAG TTA ATA CCG TTT ACT ACA TGT TGT TCA CTC AAT GAA TAT TATTCA 383 Glu Leu Ile Pro Phe Thr Thr Cys Cys Ser Leu Asn Glu Tyr Tyr Ser115 120 125 AAA ATT GTG ATT TTA ATA AAT GTT ATT TTA GAA TAT ATG ATA TCTATT 431 Lys Ile Val Ile Leu Ile Asn Val Ile Leu Glu Tyr Met Ile Ser Ile130 135 140 ATA TTA TAT AGA ATA TTG ATC GTA AAA AGA TTT AAT AAC ATT AAAGAA 479 Ile Leu Tyr Arg Ile Leu Ile Val Lys Arg Phe Asn Asn Ile Lys Glu145 150 155 TTT ATT TCA AAA GTC GTA AAT ACT GTA CTA GAA TCA TCA GGC ATATAT 527 Phe Ile Ser Lys Val Val Asn Thr Val Leu Glu Ser Ser Gly Ile Tyr160 165 170 175 TTT TGT CAG ATG CGT GTA CAT GAA CAA ATT GAA TTG GAA ATAGAT GAG 575 Phe Cys Gln Met Arg Val His Glu Gln Ile Glu Leu Glu Ile AspGlu 180 185 190 CTC ATT ATT AAT GGA TCT ATG CCT GTA CAG CTT ATG CAT TTACTT CTA 623 Leu Ile Ile Asn Gly Ser Met Pro Val Gln Leu Met His Leu LeuLeu 195 200 205 AAG GTA GCT ACC ATA ATA TTA GAG GAA ATC AAA GAA ATATAACGTATTT 672 Lys Val Ala Thr Ile Ile Leu Glu Glu Ile Lys Glu Ile 210215 220 TTTCTTTTAA ATAAATAAAA ATACTTTTTT TTTTAAACAA GGGGTGCTACCTTGTCTAAT 732 TGTATCTTGT ATTTTGGATC TGATGCAAGA TTATTAAATA ATCGTATGAAAAAGTAGTAG 792 ATATAGTTTA TATCGTTACT GGACATGATA TTATGTTTAG TTAATTCTTCTTTGGCATGA 852 ATTCTACACG TCGGANAAGG TAATGTATCT ATAATGGTAT AAAGCTT 899220 amino acids amino acid linear protein not provided 4 Asp Ile Lys SerCys Lys Cys Ser Ile Cys Ser Asp Ser Ile Thr His 1 5 10 15 His Ile TyrGlu Thr Thr Ser Cys Ile Asn Tyr Lys Ser Thr Asp Asn 20 25 30 Asp Leu MetIle Val Leu Phe Asn Leu Thr Arg Tyr Leu Met His Gly 35 40 45 Met Ile HisPro Asn Leu Ile Ser Val Lys Gly Trp Gly Pro Leu Ile 50 55 60 Gly Leu LeuThr Gly Asp Ile Gly Ile Asn Leu Lys Leu Tyr Ser Thr 65 70 75 80 Met AsnIle Asn Gly Leu Arg Tyr Gly Asp Ile Thr Leu Ser Ser Tyr 85 90 95 Asp MetSer Asn Lys Leu Val Ser Ile Ile Asn Thr Pro Ile Tyr Glu 100 105 110 LeuIle Pro Phe Thr Thr Cys Cys Ser Leu Asn Glu Tyr Tyr Ser Lys 115 120 125Ile Val Ile Leu Ile Asn Val Ile Leu Glu Tyr Met Ile Ser Ile Ile 130 135140 Leu Tyr Arg Ile Leu Ile Val Lys Arg Phe Asn Asn Ile Lys Glu Phe 145150 155 160 Ile Ser Lys Val Val Asn Thr Val Leu Glu Ser Ser Gly Ile TyrPhe 165 170 175 Cys Gln Met Arg Val His Glu Gln Ile Glu Leu Glu Ile AspGlu Leu 180 185 190 Ile Ile Asn Gly Ser Met Pro Val Gln Leu Met His LeuLeu Leu Lys 195 200 205 Val Ala Thr Ile Ile Leu Glu Glu Ile Lys Glu Ile210 215 220 129 amino acids amino acid double linear peptide YES NON-terminal Vaccinia virus Copenhagen ~23.2 %G 5 Met Phe Met Tyr Pro GluPhe Ala Arg Lys Ala Leu Ser Lys Leu Ile 1 5 10 15 Ser Lys Lys Leu AsnIle Glu Lys Val Ser Ser Lys His Gln Leu Val 20 25 30 Leu Leu Asp Tyr GlyLeu His Gly Leu Leu Pro Lys Ser Leu Tyr Leu 35 40 45 Glu Ala Ile Asn SerAsp Ile Leu Asn Val Arg Phe Phe Pro Pro Glu 50 55 60 Ile Ile Asn Val ThrAsp Ile Val Lys Ala Leu Gln Asn Ser Cys Arg 65 70 75 80 Val Asp Glu TyrLeu Lys Ala Val Ser Leu Tyr His Lys Asn Ser Leu 85 90 95 Met Val Ser GlyPro Asn Val Val Lys Leu Met Ile Glu Tyr Asn Leu 100 105 110 Leu Thr HisSer Asp Leu Glu Trp Leu Ile Asn Glu Asn Val Val Lys 115 120 125 Ala 132amino acids amino acid double linear peptide YES NO N-terminal Swinepoxvirus Kasza ~23.2 %G 6 Met Pro Ser Tyr Met Tyr Pro Lys Asn Ala Arg LysVal Ile Ser Lys 1 5 10 15 Ile Ile Ser Leu Gln Leu Asp Ile Lys Lys LeuPro Lys Lys Tyr Ile 20 25 30 Asn Thr Met Leu Glu Phe Gly Leu His Gly AsnLeu Pro Ala Cys Met 35 40 45 Tyr Lys Asp Ala Val Ser Tyr Asp Ile Asn AsnIle Arg Phe Leu Pro 50 55 60 Tyr Asn Cys Val Met Val Lys Asp Leu Ile AsnVal Ile Lys Ser Ser 65 70 75 80 Ser Val Ile Asp Thr Arg Leu His Gln SerVal Leu Lys His Arg Arg 85 90 95 Ala Leu Ile Asp Tyr Gly Asp Gln Asp IleIle Thr Leu Met Ile Ile 100 105 110 Asn Lys Leu Leu Ser Ile Asp Asp IleSer Tyr Ile Leu Asp Lys Lys 115 120 125 Ile Ile His Val 130 101 aminoacids amino acid double linear peptide YES NO C-terminal Vaccinia virusCopenhagen ~23.2 %G 7 Val Leu Asn Asp Gln Tyr Ala Lys Ile Val Ile PhePhe Asn Thr Ile 1 5 10 15 Ile Glu Tyr Ile Ile Ala Thr Ile Tyr Tyr ArgLeu Thr Val Leu Asn 20 25 30 Asn Tyr Thr Asn Val Lys His Phe Val Ser LysVal Leu His Thr Val 35 40 45 Met Glu Ala Cys Gly Val Leu Phe Ser Tyr IleLys Val Asn Asp Lys 50 55 60 Ile Glu His Glu Leu Glu Glu Met Val Asp LysGly Thr Val Pro Ser 65 70 75 80 Tyr Leu Tyr His Leu Ser Ile Asn Val IleSer Ile Ile Leu Asp Asp 85 90 95 Ile Asn Gly Thr Arg 100 100 amino acidsamino acid double linear peptide YES NO C-terminal Swinepox virus Kasza~23.2 %G 8 Ser Leu Asn Glu Tyr Tyr Ser Lys Ile Val Ile Leu Ile Asn ValIle 1 5 10 15 Leu Glu Tyr Met Ile Ser Ile Ile Leu Tyr Arg Ile Leu IleVal Lys 20 25 30 Arg Phe Asn Asn Ile Lys Glu Phe Ile Ser Lys Val Val AsnThr Val 35 40 45 Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gln Met Arg Val HisGlu Gln 50 55 60 Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile Asn Gly Ser MetPro Val 65 70 75 80 Gln Leu Met His Leu Leu Leu Lys Val Ala Thr Ile IleLeu Glu Glu 85 90 95 Ile Lys Glu Ile 100 102 base pairs nucleic aciddouble circular DNA (genomic) NO NO Plasmid 520-17.5 (Junction A) FrancoA Trach, Kathleen Hoch, James AFerrari Sequence Analysis of the spo0BLocus Revels a Polycistronic Transcription Unit J. Bacteriol. 161 2556-562 Feb.-1985 9 CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTATACCATTATAG ATACATTACC 60 TTGTCCGACG TGTAGAATTC ATGCCAAAGA AGAATTAACT AA102 102 base pairs nucleic acid double circular DNA (genomic) NO NOPlasmid 520-17.5 (Junction B) CDS 85..99 /codon_start= 85 /function=“Translational start of hybrid protein” /product= “N-terminal peptide”/number= 1 /standard_name= “Translation of synthetic DNA sequence” CDS100..102 experimental /partial /codon_start= 100 /function= “markerenzyme” /product= “Beta-Galactosidase” /evidence= EXPERIMENTAL /gene=“lacZ” /number= 2 /citation= ([1]) Franco A Trach, Kathleen Hoch, JamesAFerrari Seqquence Analysis of the spo0B Locus Reveals a PolycistronicTranscription Unit J. Bacteriol. 161 2 556-562 Feb.-1985 10 GTAGTCGACTCTAGAAAAAA TTGAAAAACT ATTCTAATTT ATTGCACGGA GATCTTTTTT 60 TTTTTTTTTTTTTTTGGCAT ATAA ATG AAT TCG GAT CCC GTC 102 Met Asn Ser Asp Pro Val 1 55 amino acids amino acid linear peptide not provided 11 Met Asn Ser AspPro 1 5 1 amino acids amino acid linear peptide not provided 12 Val 1103 base pairs nucleic acid double circular DNA (genomic) NO NO Plasmid520-17.5 (Junction C) CDS 1..72 experimental /partial /codon_start= 1/function= “marker enzyme” /product= “Beta-galactosidase” /evidence=EXPERIMENTAL /gene= “lacZ” /number= 1 /citation= ([1]) CDS 73..78experimental /codon_start= 73 /function= “Translational finish of hybridprotein” /product= “C-terminal peptide” /evidence= EXPERIMENTAL /number=2 /standard_name= “Translation of synthetic DNA sequence” Franco ATrach, Kathleen Hoch, James AFerrari Seqquence Analysis of the spo0BLocus Reveals a Polycistronic Transcription Unit J. Bacteriol. 161 2556-562 Feb.-1985 13 AGC CCG TCA GTA TCG GCG GAA ATC CAG CTG AGC GCC GGTCGC TAC CAT 48 Ser Pro Ser Val Ser Ala Glu Ile Gln Leu Ser Ala Gly ArgTyr His 1 5 10 15 TAC CAG TTG GTC TGG TGT CAA AAA GAT CCA TAATTAATTAACCCGGGTCG 98 Tyr Gln Leu Val Trp Cys Gln Lys Asp Pro 20 25 AAGAC 103 24amino acids amino acid linear peptide not provided 14 Ser Pro Ser ValSer Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His 1 5 10 15 Tyr Gln LeuVal Trp Cys Gln Lys 20 2 amino acids amino acid linear peptide notprovided 15 Asp Pro 1 48 base pairs nucleic acid double circular DNA(genomic) NO NO Plasmid 520-17.5 (Junction D) 16 AGATCCCCGG GCGAGCTCGAATTCGTAATC ATGGTCATAG CTGTTTCC 48 57 base pairs nucleic acid doublecircular DNA (genomic) NO NO Plasmid 538-46.26 (Junction A) 17CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTAT ACCATTATAG ATACATT 57 102base pairs nucleic acid double circular DNA (genomic) NO NO Plasmid538-46.16 (Junction B) CDS 91..102 experimental /partial /codon_start=91 /function= “marker enzyme” /product= “Beta-Galactosidase” /evidence=EXPERIMENTAL /gene= “lacZ” /number= 2 /citation= ([1]) CDS 76..90/partial /codon_start= 76 /function= “Translational start of hybridprotein” /product= “N-terminal peptide” /number= 1 /standard_name=“Translation of synthetic DNA sequence” Franco A Trach, Kathleen Hoch,James AFerrari Seqquence Analysis of the spo0B Locus Reveals aPolycistronic Transcription Unit J. Bacteriol. 161 2 556-562 Feb.-198518 AAGCTGGTAG ATTTCCATGT AGGGCCGCCT GCAGGTCGAC TCTAGAATTT CATTTTGTTT 60TTTTCTATGC TATAA ATG AAT TCG GAT CCC GTC GTT TTA CAA 102 Met Asn Ser AspPro Val Val Leu Gln 1 5 5 amino acids amino acid linear peptide notprovided 19 Met Asn Ser Asp Pro 1 5 4 amino acids amino acid linearpeptide not provided 20 Val Val Leu Gln 1 206 base pairs nucleic aciddouble circular DNA (genomic) NO NO Plasmid 538-46.16 (Junction C) CDS1..63 experimental /partial /codon_start= 1 /function= “marker enzyme”/product= “Beta-galactosidase” /evidence= EXPERIMENTAL /number= 1/citation= ([1]) CDS 64..69 experimental /codon_start= 64 /function=“Translational finish of hybrid protein” /product= “C-terminal peptide”/evidence= EXPERIMENTAL /standard_name= “Translation of synthetic DNAsequence” CDS 177..185 experimental /codon_start= 177 /function=“Translational start of hybrid protein” /product= “N-terminal peptide”/evidence= EXPERIMENTAL /standard_name= “Translation of synthetic DNAsequence” CDS 186..206 experimental /partial /codon_start= 186/function= “glycoprotein” /product= “PRV gp50” /evidence= EXPERIMENTAL/gene= “gp50” /number= 3 /citation= ([2]) Franco A Trach, Kathleen Hoch,James AFerrari Seqquence Analysis of the spo0B Locus Reveals aPolycistronic Transcription Unit J. Bacteriol. 161 2 556-562 Feb.-1985Erik A Timmins, James G Armentrout, Marty A Marchioli, Carmine C Jr.Yancy, Robert J Post, Leonard EPetrovskis DNA Sequence of the Gene forPseudorabies Virus gp50, a Glycoprotein without N-Linked GlycosylationJ. Virol. 59 2 216-223 Aug.-1986 21 GTA TCG GCG GAA ATC CAG CTG AGC GCCGGT CGC TAC CAT TAC CAG TTG 48 Val Ser Ala Glu Ile Gln Leu Ser Ala GlyArg Tyr His Tyr Gln Leu 1 5 10 15 GTC TGG TGT CAA AAA GAT CCA TAATTAATTAACCCGGCCGC CTGCAGGTCG 99 Val Trp Cys Gln Lys Asp Pro 20 ACTCTAGAAAAAATTGAAAA ACTATTCTAA TTTATTGCAC GGAGATCTTT TTTTTTTTTT 159 TTTTTTTTGGCATATAA ATG AAT TCG CTC GCA GCG CTA TTG GCG GCG 206 Met Asn Ser Leu AlaAla Leu Leu Ala Ala 1 1 5 21 amino acids amino acid linear peptide notprovided 22 Val Ser Ala Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr GlnLeu 1 5 10 15 Val Trp Cys Gln Lys 20 2 amino acids amino acid linearpeptide not provided 23 Asp Pro 1 3 amino acids amino acid linearpeptide not provided 24 Met Asn Ser 1 7 amino acids amino acid linearpeptide not provided 25 Leu Ala Ala Leu Leu Ala Ala 1 5 101 base pairsnucleic acid double circular DNA (genomic) NO NO Plasmid 538-46.16(Junction D) CDS 1..15 /partial /codon_start= 1 /function=“glycoprotein” /product= “PRV gp63” /gene= “gp63” /number= 1 /citation=([1]) Erik A Timmins, James G Post, Lenoard EPetrovskis Use ofLambda-gt11 To Isolate Genes for two Pseudorabies Virus Glycoproteinswith homology to Herpes Simplex Virus and Varicella-Zoster VirusGlycoproteins J. Virol. 60 1 185-193 Oct.-1986 26 CGC GTG CAC CAC GAGGGACTCTAGA GGATCCATAA TTAATTAATT AATTTTTATC 55 Arg Val His His Glu 1 5CCGGGTCGAC CTGCAGGCGG CCGGGTCGAC CTGCAGGCGG CCAGAC 101 5 amino acidsamino acid linear peptide not provided 27 Arg Val His His Glu 1 5 57base pairs nucleic acid double circular DNA (genomic) NO NO Plasmid538-46.16 (Junction E) 28 AGATCCCCGG GCGAGCTCGA ATTCGTAATC ATGGTCATAGCTGTTTCCTG TGTGAAA 57 1907 base pairs nucleic acid double linear cDNA tomRNA NO NO Newcastle disease virus B1 137-23.803 (PSY1142) ~50% %G CDS92..1822 /codon_start= 92 /product= “NDV heamagglutinin-Neuraminidase”/gene= “HN” /number= 1 29 ACGGGTAGAA CGGTAAGAGA GGCCGCCCCT CAATTGCGAGCCAGACTTCA CAACCTCCGT 60 TCTACCGCTT CACCGACAAC AGTCCTCAAT C ATG GAC CGCGCC GTT AGC CAA 112 Met Asp Arg Ala Val Ser Gln 1 5 GTT GCG TTA GAG AATGAT GAA AGA GAG GCA AAA AAT ACA TGG CGC TTG 160 Val Ala Leu Glu Asn AspGlu Arg Glu Ala Lys Asn Thr Trp Arg Leu 10 15 20 ATA TTC CGG ATT GCA ATCTTA TTC TTA ACA GTA GTG ACC TTG GCT ATA 208 Ile Phe Arg Ile Ala Ile LeuPhe Leu Thr Val Val Thr Leu Ala Ile 25 30 35 TCT GTA GCC TCC CTT TTA TATAGC ATG GGG GCT AGC ACA CCT AGC GAT 256 Ser Val Ala Ser Leu Leu Tyr SerMet Gly Ala Ser Thr Pro Ser Asp 40 45 50 55 CTT GTA GGC ATA CCG ACT AGGATT TCC AGG GCA GAA GAA AAG ATT ACA 304 Leu Val Gly Ile Pro Thr Arg IleSer Arg Ala Glu Glu Lys Ile Thr 60 65 70 TCT ACA CTT GGT TCC AAT CAA GATGTA GTA GAT AGG ATA TAT AAG CAA 352 Ser Thr Leu Gly Ser Asn Gln Asp ValVal Asp Arg Ile Tyr Lys Gln 75 80 85 GTG GCC CTT GAG TCT CCA TTG GCA TTGTTA AAT ACT GAG ACC ACA ATT 400 Val Ala Leu Glu Ser Pro Leu Ala Leu LeuAsn Thr Glu Thr Thr Ile 90 95 100 ATG AAC GCA ATA ACA TCT CTC TCT TATCAG ATT AAT GGA GCT GCA AAC 448 Met Asn Ala Ile Thr Ser Leu Ser Tyr GlnIle Asn Gly Ala Ala Asn 105 110 115 AAC AGC GGG TGG GGG GCA CCT ATT CATGAC CCA GAT TAT ATA GGG GGG 496 Asn Ser Gly Trp Gly Ala Pro Ile His AspPro Asp Tyr Ile Gly Gly 120 125 130 135 ATA GGC AAA GAA CTC ATT GTA GATGAT GCT AGT GAT GTC ACA TCA TTC 544 Ile Gly Lys Glu Leu Ile Val Asp AspAla Ser Asp Val Thr Ser Phe 140 145 150 TAT CCC TCT GCA TTT CAA GAA CATCTG AAT TTT ATC CCG GCG CCT ACT 592 Tyr Pro Ser Ala Phe Gln Glu His LeuAsn Phe Ile Pro Ala Pro Thr 155 160 165 ACA GGA TCA GGT TGC ACT CGA ATACCC TCA TTT GAC ATG AGT GCT ACC 640 Thr Gly Ser Gly Cys Thr Arg Ile ProSer Phe Asp Met Ser Ala Thr 170 175 180 CAT TAC TGC TAC ACC CAT AAT GTAATA TTG TCT GGA TGC AGA GAT CAC 688 His Tyr Cys Tyr Thr His Asn Val IleLeu Ser Gly Cys Arg Asp His 185 190 195 TCA CAC TCA CAT CAG TAT TTA GCACTT GGT GTG CTC CGG ACA TCT GCA 736 Ser His Ser His Gln Tyr Leu Ala LeuGly Val Leu Arg Thr Ser Ala 200 205 210 215 ACA GGG AGG GTA TTC TTT TCTACT CTG CGT TCC ATC AAC CTG GAC GAC 784 Thr Gly Arg Val Phe Phe Ser ThrLeu Arg Ser Ile Asn Leu Asp Asp 220 225 230 ACC CAA AAT CGG AAG TCT TGCAGT GTG AGT GCA ACT CCC CTG GGT TGT 832 Thr Gln Asn Arg Lys Ser Cys SerVal Ser Ala Thr Pro Leu Gly Cys 235 240 245 GAT ATG CTG TGC TCG AAA GCCACG GAG ACA GAG GAA GAA GAT TAT AAC 880 Asp Met Leu Cys Ser Lys Ala ThrGlu Thr Glu Glu Glu Asp Tyr Asn 250 255 260 TCA GCT GTC CCT ACG CGG ATGGTA CAT GGG AGG TTA GGG TTC GAC GGC 928 Ser Ala Val Pro Thr Arg Met ValHis Gly Arg Leu Gly Phe Asp Gly 265 270 275 CAA TAT CAC GAA AAG GAC CTAGAT GTC ACA ACA TTA TTC GGG GAC TGG 976 Gln Tyr His Glu Lys Asp Leu AspVal Thr Thr Leu Phe Gly Asp Trp 280 285 290 295 GTG GCC AAC TAC CCA GGAGTA GGG GGT GGA TCT TTT ATT GAC AGC CGC 1024 Val Ala Asn Tyr Pro Gly ValGly Gly Gly Ser Phe Ile Asp Ser Arg 300 305 310 GTG TGG TTC TCA GTC TACGGA GGG TTA AAA CCC AAT ACA CCC AGT GAC 1072 Val Trp Phe Ser Val Tyr GlyGly Leu Lys Pro Asn Thr Pro Ser Asp 315 320 325 ACT GTA CAG GAA GGG AAATAT GTG ATA TAC AAG CGA TAC AAT GAC ACA 1120 Thr Val Gln Glu Gly Lys TyrVal Ile Tyr Lys Arg Tyr Asn Asp Thr 330 335 340 TGC CCA GAT GAG CAA GACTAC CAG ATT CGA ATG GCC AAG TCT TCG TAT 1168 Cys Pro Asp Glu Gln Asp TyrGln Ile Arg Met Ala Lys Ser Ser Tyr 345 350 355 AAG CCT GGA CGG TTT GGTGGG AAA CGC ATA CAG CAG GCT ATC TTA TCT 1216 Lys Pro Gly Arg Phe Gly GlyLys Arg Ile Gln Gln Ala Ile Leu Ser 360 365 370 375 ATC AAA GTG TCA ACATCC TTA GGC GAA GAC CCG GTA CTG ACT GTA CCG 1264 Ile Lys Val Ser Thr SerLeu Gly Glu Asp Pro Val Leu Thr Val Pro 380 385 390 CCC AAC ACA GTC ACACTC ATG GGG GCC GAA GGC AGA ATT CTC ACA GTA 1312 Pro Asn Thr Val Thr LeuMet Gly Ala Glu Gly Arg Ile Leu Thr Val 395 400 405 GGG ACA TCC CAT TTCTTG TAT CAG CGA GGG TCA TCA TAC TTC TCT CCC 1360 Gly Thr Ser His Phe LeuTyr Gln Arg Gly Ser Ser Tyr Phe Ser Pro 410 415 420 GCG TTA TTA TAT CCTATG ACA GTC AGC AAC AAA ACA GCC ACT CTT CAT 1408 Ala Leu Leu Tyr Pro MetThr Val Ser Asn Lys Thr Ala Thr Leu His 425 430 435 AGT CCT TAT ACA TTCAAT GCC TTC ACT CGG CCA GGT AGT ATC CCT TGC 1456 Ser Pro Tyr Thr Phe AsnAla Phe Thr Arg Pro Gly Ser Ile Pro Cys 440 445 450 455 CAG GCT TCA GCAAGA TGC CCC AAC TCA TGT GTT ACT GGA GTC TAT ACA 1504 Gln Ala Ser Ala ArgCys Pro Asn Ser Cys Val Thr Gly Val Tyr Thr 460 465 470 GAT CCA TAT CCCCTA ATC TTC TAT AGA AAC CAC ACC TTG CGA GGG GTA 1552 Asp Pro Tyr Pro LeuIle Phe Tyr Arg Asn His Thr Leu Arg Gly Val 475 480 485 TTC GGG ACA ATGCTT GAT GGT GAA CAA GCA AGA CTT AAC CCT GCG TCT 1600 Phe Gly Thr Met LeuAsp Gly Glu Gln Ala Arg Leu Asn Pro Ala Ser 490 495 500 GCA GTA TTC GATAGC ACA TCC CGC AGT CGC ATA ACT CGA GTG AGT TCA 1648 Ala Val Phe Asp SerThr Ser Arg Ser Arg Ile Thr Arg Val Ser Ser 505 510 515 AGC AGC ATC AAAGCA GCA TAC ACA ACA TCA ACT TGT TTT AAA GTG GTC 1696 Ser Ser Ile Lys AlaAla Tyr Thr Thr Ser Thr Cys Phe Lys Val Val 520 525 530 535 AAG ACC AATAAG ACC TAT TGT CTC AGC ATT GCT GAA ATA TCT AAT ACT 1744 Lys Thr Asn LysThr Tyr Cys Leu Ser Ile Ala Glu Ile Ser Asn Thr 540 545 550 CTC TTC GGAGAA TTC AGA ATC GTC CCG TTA CTA GTT GAG ATC CTC AAA 1792 Leu Phe Gly GluPhe Arg Ile Val Pro Leu Leu Val Glu Ile Leu Lys 555 560 565 GAT GAC GGGGTT AGA GAA GCC AGG TCT GGC TAGTTGAGTC AACTATGAAA 1842 Asp Asp Gly ValArg Glu Ala Arg Ser Gly 570 575 GAGTTGGAAA GATGGCATTG TATCACCTATCTTCTGCGAC ATCAAGAATC AAACCGAATG 1902 CCGGC 1907 577 amino acids aminoacid linear protein not provided 30 Met Asp Arg Ala Val Ser Gln Val AlaLeu Glu Asn Asp Glu Arg Glu 1 5 10 15 Ala Lys Asn Thr Trp Arg Leu IlePhe Arg Ile Ala Ile Leu Phe Leu 20 25 30 Thr Val Val Thr Leu Ala Ile SerVal Ala Ser Leu Leu Tyr Ser Met 35 40 45 Gly Ala Ser Thr Pro Ser Asp LeuVal Gly Ile Pro Thr Arg Ile Ser 50 55 60 Arg Ala Glu Glu Lys Ile Thr SerThr Leu Gly Ser Asn Gln Asp Val 65 70 75 80 Val Asp Arg Ile Tyr Lys GlnVal Ala Leu Glu Ser Pro Leu Ala Leu 85 90 95 Leu Asn Thr Glu Thr Thr IleMet Asn Ala Ile Thr Ser Leu Ser Tyr 100 105 110 Gln Ile Asn Gly Ala AlaAsn Asn Ser Gly Trp Gly Ala Pro Ile His 115 120 125 Asp Pro Asp Tyr IleGly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp 130 135 140 Ala Ser Asp ValThr Ser Phe Tyr Pro Ser Ala Phe Gln Glu His Leu 145 150 155 160 Asn PheIle Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170 175 SerPhe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile 180 185 190Leu Ser Gly Cys Arg Asp His Ser His Ser His Gln Tyr Leu Ala Leu 195 200205 Gly Val Leu Arg Thr Ser Ala Thr Gly Arg Val Phe Phe Ser Thr Leu 210215 220 Arg Ser Ile Asn Leu Asp Asp Thr Gln Asn Arg Lys Ser Cys Ser Val225 230 235 240 Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys AlaThr Glu 245 250 255 Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr ArgMet Val His 260 265 270 Gly Arg Leu Gly Phe Asp Gly Gln Tyr His Glu LysAsp Leu Asp Val 275 280 285 Thr Thr Leu Phe Gly Asp Trp Val Ala Asn TyrPro Gly Val Gly Gly 290 295 300 Gly Ser Phe Ile Asp Ser Arg Val Trp PheSer Val Tyr Gly Gly Leu 305 310 315 320 Lys Pro Asn Thr Pro Ser Asp ThrVal Gln Glu Gly Lys Tyr Val Ile 325 330 335 Tyr Lys Arg Tyr Asn Asp ThrCys Pro Asp Glu Gln Asp Tyr Gln Ile 340 345 350 Arg Met Ala Lys Ser SerTyr Lys Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365 Ile Gln Gln Ala IleLeu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu 370 375 380 Asp Pro Val LeuThr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala 385 390 395 400 Glu GlyArg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gln Arg 405 410 415 GlySer Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser 420 425 430Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr 435 440445 Arg Pro Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser 450455 460 Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg465 470 475 480 Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp GlyGlu Gln 485 490 495 Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser ThrSer Arg Ser 500 505 510 Arg Ile Thr Arg Val Ser Ser Ser Ser Ile Lys AlaAla Tyr Thr Thr 515 520 525 Ser Thr Cys Phe Lys Val Val Lys Thr Asn LysThr Tyr Cys Leu Ser 530 535 540 Ile Ala Glu Ile Ser Asn Thr Leu Phe GlyGlu Phe Arg Ile Val Pro 545 550 555 560 Leu Leu Val Glu Ile Leu Lys AspAsp Gly Val Arg Glu Ala Arg Ser 565 570 575 Gly 57 base pairs nucleicacid double circular DNA (genomic) NO NO Plasmid 538-46.26 (Junction A)31 CACATACGAT TTAGGTGACA CTATAGAATA CAAGCTTTAT ACCATTATAG ATACATT 57 108base pairs nucleic acid double circular DNA (genomic) NO NO Plasmid538-46.26 (Junction B) exon 88..102 /codon_start= 88 /function=“Translational start of hybrid protein” /product= “N-terminal peptide”/number= 1 /standard_name= “Translation of synthetic DNA sequence” CDS103..108 experimental /partial /codon_start= 103 /product= “NDVHeamagglutinin-Neuraminidase” /evidence= EXPERIMENTAL /gene= “HN”/number= 2 32 CATGTAGTCG ACTCTAGAAA AAATTGAAAA ACTATTCTAA TTTATTGCACGGAGATCTTT 60 TTTTTTTTTT TTTTTTTTGG CATATAAATG AATTCGGATC CG GAC CGC 108Asp Arg 1 2 amino acids amino acid linear peptide not provided 33 AspArg 1 108 base pairs nucleic acid double circular DNA (genomic) NO NOPlasmid 538-46.26 (Junction C) CDS 70..84 /codon_start= 70 /function=“Translational start of hybrid protein” /product= “N-terminal peptide”/number= 1 /standard_name= “Translation of synthetic DNA sequence” CDS85..108 experimental /partial /codon_start= 85 /function= “markerenzyme” /product= “Beta-galactosidase” /evidence= EXPERIMENTAL /gene=“lacZ” /number= 2 /citation= ([1]) Franco A Trach, Kathleen Hoch, JamesAFerrari Sequence Analysis of the spo0B Locus Reveals a PolycistronicTranscription Unit J. Bacteriol. 161 2 556-562 Feb.-1985 34 TGCGACATCAAGAATCAAAC CGAATGCCCT CGACTCTAGA ATTTCATTTT GTTTTTTTCT 60 ATGCTATAA ATGAAT TCG GAT CCC GTC GTT TTA CAA CGT CGT GAC TGG 108 Met Asn Ser Asp ProVal Val Leu Gln Arg Arg Asp Trp 1 5 10 5 amino acids amino acid linearpeptide not provided 35 Met Asn Ser Asp Pro 1 5 8 amino acids amino acidlinear peptide not provided 36 Val Val Leu Gln Arg Arg Asp Trp 1 5 108base pairs nucleic acid double circular DNA (genomic) NO NO Plasmid538-46.26 CDS 1..54 experimental /partial /codon_start= 1 /function=“marker enzyme” /product= “Beta-galactosidase” /evidence= EXPERIMENTAL/gene= “lacZ” /number= 1 /citation= ([1]) CDS 55..63 experimental/codon_start= 55 /function= “Translational finish of hybrid protein”/product= “C-terminal peptide” /evidence= EXPERIMENTAL /number= 2/standard_name= “Translation of synthetic DNA sequence” 37 GAA ATC CAGCTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gln LeuSer Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 15 CAA AAA GATCCA TAATTAATTA ACCCGGGTCG AGGGTCGAAG ACCAAATTCT 100 Gln Lys Asp Pro 20AACATGGT 108 18 amino acids amino acid linear protein not provided 38Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 1015 Gln Lys 2 amino acids amino acid linear peptide not provided 39 AspPro 1 57 base pairs nucleic acid double circular DNA (genomic) NO NOPlasmid 538-46.26 (Junction E) 40 AGATCCCCGG GCGAGCTCGA ATTCGTAATCATGGTCATAG CTGTTTCCTG TGTGAAA 57 27 base pairs nucleic acid singlelinear DNA (genomic) N N Pseudorabies virus Synthetic oligonucleotideprimer 41 CGCGAATTCG CTCGCAGCGC TATTGGC 27 19 base pairs nucleic acidsingle linear DNA (genomic) N N Pseudorabies virus Syntheticoligonucleotide primer 42 GTAGGAGTGG CTGCTGAAG 19 70 base pairs nucleicacid double linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 43 AAAAATTGAA AAACTATTCT AATTTATTGC ACGGAGATCTTTTTTTTTTT TTTTTTTTTG 60 GCATATAAAT 70 74 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2%G 44 TTTTTTTTTT TTTTTTTTTT GGCATATAAA TAGATCTGTA TCCTAAAATT GAATTGTAAT60 TATCGATAAT AAAT 74 37 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 45GTATCCTAAA ATTGAATTGT AATTATCGAT AATAAAT 37 41 base pairs nucleic aciddouble linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 46 CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA T 41 60base pairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G 47 CACATACGAT TTAGGTGACA CTATAGAATACAAGCTTTGA GTCTATTGGT TATTTATACG 60 123 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2%G CDS 100..123 48 TGAATATATA GCAAATAAAG GAAAAATTGT TATCGTTGCTGCATTAGATG GAACATAGGT 60 CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAA ATGAAT TCG GAT CCC 114 Met Asn Ser Asp Pro 1 5 GTC GTT TTA 123 Val Val Leu8 amino acids amino acid linear peptide not provided 49 Met Asn Ser AspPro Val Val Leu 1 5 132 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS1..63 50 GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGG TGT48 Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 510 15 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG ACCTATGAAC GTAAACCATT 100Gln Lys Asp Pro 20 TGGTAATATT CTTAATCTTA TACCATTATC GG 132 20 aminoacids amino acid linear protein not provided 51 Glu Ile Gln Leu Ser AlaGly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 15 Gln Lys Asp Pro 20 66base pairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G 52 TCTACTATTG TATATATAGG ATCCCCGGGCGAGCTCGAAT TCGTAATCAT GGTCATAGCT 60 GTTTCC 66 51 base pairs nucleic aciddouble linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 53 ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCGCCCGGGGATC T 51 104 base pairs nucleic acid double linear DNA (genomic)NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS 81..104 54AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT 60TGTTTTTTTC TATGCTATAA ATG AAT TCG GAT CCC GTC GTT TTA 104 Met Asn SerAsp Pro Val Val Leu 1 5 8 amino acids amino acid linear peptide notprovided 55 Met Asn Ser Asp Pro Val Val Leu 1 5 150 base pairs nucleicacid double linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G CDS 1..63 CDS 130..150 56 GAA ATC CAG CTG AGC GCC GGTCGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gln Leu Ser Ala Gly ArgTyr His Tyr Gln Leu Val Trp Cys 1 5 10 15 CAA AAA GAT CCA TAATTAATTAACCCGGTCGA CTCTAGAAAG ATCTGTATCC 100 Gln Lys Asp Pro 20 TAAAATTGAATTGTAATTAT CGATAATAA ATG AAT TCC GGC ATG GCC TCG 150 Met Asn Ser Gly MetAla Ser 1 5 20 amino acids amino acid linear peptide not provided 57 GluIle Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 15Gln Lys Asp Pro 20 7 amino acids amino acid linear peptide not provided58 Met Asn Ser Gly Met Ala Ser 1 5 109 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2%G 59 CCATGCTCTA GAGGATCCCC GGGCGAGCTC GAATTCGGAT CCATAATTAA TTAATTAATT60 TTTATCCCGG GTCGACCGGG TCGACCTGCA GCCTACATGG AAATCTACC 109 51 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G 60 TAATGTATCT ATAATGGTAT AAAGCTTGTATTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 61ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 104 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G CDS 81..104 62 AAATATATAA ATACCATGTTAGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT 60 TGTTTTTTTC TATGCTATAA ATGAAT TCG GAT CCC GTC GTT TTA 104 Met Asn Ser Asp Pro Val Val Leu 1 5 8amino acids amino acid linear peptide not provided 63 Met Asn Ser AspPro Val Val Leu 1 5 182 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS1..63 CDS 156..182 64 GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAGTTG GTC TGG TGT 48 Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln LeuVal Trp Cys 1 5 10 15 CAA AAA GAT CCA TAATTAATTA ACCCGGTCGA CTCTAGAAAAAATTGAAAAA 100 Gln Lys Asp Pro 20 CTATTCTAAT TTATTGCACG GAGATCTTTTTTTTTTTTTT TTTTTTGGCA TATAA ATG 158 Met 1 AAT TCC GGC ATG GCC TCG CTCGCG 182 Asn Ser Gly Met Ala Ser Leu Ala 5 20 amino acids amino acidlinear peptide not provided 65 Glu Ile Gln Leu Ser Ala Gly Arg Tyr HisTyr Gln Leu Val Trp Cys 1 5 10 15 Gln Lys Asp Pro 20 9 amino acids aminoacid linear peptide not provided 66 Met Asn Ser Gly Met Ala Ser Leu Ala1 5 109 base pairs nucleic acid double linear DNA (genomic) NO NOSwinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 67 CCATGCTCTAGAGGATCCCC GGGCGAGCTC GAATTCGGAT CCATAATTAA TTAATTAATT 60 TTTATCCCGGGTCGACCGGG TCGACCTGCA GCCTACATGG AAATCTACC 109 51 base pairs nucleicacid double linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 68 TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTGTCACCTAAAT C 51 51 base pairs nucleic acid double linear DNA (genomic)NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 69 ACAGGAAACAGCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 104 base pairs nucleicacid double linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G CDS 81..104 70 AAATATATAA ATACCATGTT AGAATTTGGTCTGCTGCAGG TCGACTCTAG AATTTCATTT 60 TGTTTTTTTC TATGCTATAA ATG AAT TCGGAT CCC GTC GTT TTA 104 Met Asn Ser Asp Pro Val Val Leu 1 5 8 aminoacids amino acid linear peptide not provided 71 Met Asn Ser Asp Pro ValVal Leu 1 5 180 base pairs nucleic acid double linear DNA (genomic) NONO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS 1..63 CDS160..180 72 GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGGTGT 48 Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 15 10 15 CAA AAA GAT CCA TAATTAATTA ACCCGGTCGA CTCTAGATTT TTTTTTTTTT 100Gln Lys Asp Pro 20 TTTTTTTGGC ATATAAATAG ATCTGTATCC TAAAATTGAATTGTAATTAT CGATAATAA 159 ATG AAT TCC GGC ATG GCC TCG 180 Met Asn Ser GlyMet Ala Ser 1 5 20 amino acids amino acid linear peptide not provided 73Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 1015 Gln Lys Asp Pro 20 7 amino acids amino acid linear peptide notprovided 74 Met Asn Ser Gly Met Ala Ser 1 5 109 base pairs nucleic aciddouble linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 75 CCATGCTCTA GAGGATCCCC GGGCGAGCTC GAATTCGGATCCATAATTAA TTAATTAATT 60 TTTATCCCGG GTCGACCGGG TCGACCTGCA GCCTACATGGAAATCTACC 109 51 base pairs nucleic acid double linear DNA (genomic) NONO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 76 TAATGTATCTATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleicacid double linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 77 ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCGCCCGGGGATC T 51 117 base pairs nucleic acid double linear DNA (genomic)NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS 94..117 78GGTCTGCTGC AGGTCGACTC TAGAAAAAAT TGAAAAACTA TTCTAATTTA TTGCACGGAG 60ATCTTTTTTT TTTTTTTTTT TTTTGGCATA TAA ATG AAT TCC GGC TTC AGT AAC ATA 117Met Asn Ser Gly Phe Ser Asn Ile 1 5 8 8 amino acids amino acid linearpeptide not provided 79 Met Asn Ser Gly Phe Ser Asn Ile 1 5 126 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G CDS 103..126 80 CGCAACATAC CTAACTGCTTCATTTCTGAT CCATAATTAA TTAATTTTTA TCCCGGCGCG 60 CCTCGACTCT AGAATTTCATTTTGTTTTTT TCTATGCTAT AA ATG AAT TCG GAT 114 Met Asn Ser Asp 1 CCC GTCGTT TTA 126 Pro Val Val Leu 5 8 amino acids amino acid linear peptidenot provided 81 Met Asn Ser Asp Pro Val Val Leu 1 5 96 base pairsnucleic acid double linear DNA (genomic) NO NO Swinepox virus KaszaS-SPV-001 515-85.1 ~23.2 %G CDS 1..63 82 GAA ATC CAG CTG AGC GCC GGT CGCTAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gln Leu Ser Ala Gly Arg TyrHis Tyr Gln Leu Val Trp Cys 1 5 10 15 CAA AAA GAT CCA TAATTAATTAACCCGGGTCG ACCTGCAGCC TACATG 96 Gln Lys Asp Pro 20 20 amino acids aminoacid linear peptide not provided 83 Glu Ile Gln Leu Ser Ala Gly Arg TyrHis Tyr Gln Leu Val Trp Cys 1 5 10 15 Gln Lys Asp Pro 20 51 base pairsnucleic acid double linear DNA (genomic) NO NO Swinepox virus KaszaS-SPV-001 515-85.1 ~23.2 %G 84 TAATGTATCT ATAATGGTAT AAAGCTTGTATTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 85ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 124 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G CDS 104..124 86 GTATAGCGGC CGCCTGCAGGTCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAATTGAATTGTAA TTATCGATAA TAA ATG AAT TCG CTA CTT 118 Met Asn Ser Leu Leu 15 GGA ACT 124 Gly Thr 7 amino acids amino acid linear peptide notprovided 87 Met Asn Ser Leu Leu Gly Thr 1 5 126 base pairs nucleic aciddouble linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G CDS 1..12 CDS 103..126 88 ATA AAA ATG TGATTAAGTCTGAATGTGGA TCCATAATTA ATTAATTTTT 49 Ile Lys Met 1 ATCCCGGCGC GCCTCGACTCTAGAATTTCA TTTTGTTTTT TTCTATGCTA TAA ATG 105 Met 1 AAT TCG GAT CCC GTCGTT TTA 126 Asn Ser Asp Pro Val Val Leu 5 3 amino acids amino acidlinear peptide not provided 89 Ile Lys Met 1 8 amino acids amino acidlinear peptide not provided 90 Met Asn Ser Asp Pro Val Val Leu 1 5 116base pairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G CDS 1..63 91 GAA ATC CAG CTG AGC GCCGGT CGC TAC CAT TAC CAG TTG GTC TGG TGT 48 Glu Ile Gln Leu Ser Ala GlyArg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 15 CAA AAA GAT CCA TAATTAATTAACCCGGGTCG AGGCGCGCCG GGTCGACCTG 100 Gln Lys Asp Pro 20 CAGGCGGCCGCTATAC 116 20 amino acids amino acid linear peptide not provided 92 GluIle Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 1 5 10 15Gln Lys Asp Pro 20 51 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 93TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 515-85.1 ~23.2 %G 94 ACAGGAAACA GCTATGACCA TGATTACGAATTCGAGCTCG CCCGGGGATC T 51 124 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS104..124 95 GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTTTGGCATATAA 60 ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAA ATG AATTCC CCT GCC 118 Met Asn Ser Pro Ala 1 5 GCC CGG 124 Ala Arg 7 aminoacids amino acid linear peptide not provided 96 Met Asn Ser Pro Ala AlaArg 1 5 126 base pairs nucleic acid double linear DNA (genomic) NO NOSwinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS 1..36 CDS 103..12697 CTC CAG GAG CCC GCT CGC CTC GAG CGG GAT CCA TAATTAATTA ATTTTTATCC 53Leu Gln Glu Pro Ala Arg Leu Glu Arg Asp Pro 1 5 10 CGGCGCGCCT CGACTCTAGAATTTCATTTT GTTTTTTTCT ATGCTATAA ATG AAT 108 Met Asn 1 TCG GAT CCC GTCGTT TTA 126 Ser Asp Pro Val Val Leu 5 11 amino acids amino acid linearpeptide not provided 98 Leu Gln Glu Pro Ala Arg Leu Glu Arg Asp Pro 1 510 8 amino acids amino acid linear peptide not provided 99 Met Asn SerAsp Pro Val Val Leu 1 5 116 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS1..63 100 GAA ATC CAG CTG AGC GCC GGT CGC TAC CAT TAC CAG TTG GTC TGGTGT 48 Glu Ile Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys 15 10 15 CAA AAA GAT CCA TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG 100Gln Lys Asp Pro 20 CAGGCGGCCG CTATAC 116 20 amino acids amino acidlinear peptide not provided 101 Glu Ile Gln Leu Ser Ala Gly Arg Tyr HisTyr Gln Leu Val Trp Cys 1 5 10 15 Gln Lys Asp Pro 20 51 base pairsnucleic acid double linear DNA (genomic) NO NO Swinepox virus KaszaS-SPV-001 515-85.1 ~23.2 %G 102 TAATGTATCT ATAATGGTAT AAAGCTTGTATTCTATAGTG TCACCTAAAT C 51 30 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 103CCGAATTCCG GCTTCAGTAA CATAGGATCG 30 20 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2%G 104 GTACCCATAC TGGTCGTGGC 20 26 base pairs nucleic acid double linearDNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 105CCGGAATTCG CTACTTGGAA CTCTGG 26 20 base pairs nucleic acid double linearDNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 106CATTGTCCCG AGACGGACAG 20 19 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 107CGCGATCCAA CTATCGGTG 19 26 base pairs nucleic acid double linear DNA(genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 108GCGGATCCAC ATTCAGACTT AATCAC 26 30 base pairs nucleic acid double linearDNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 109ATGAATTCCC CTGCCGCCCG GACCGGCACC 30 30 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2%G 110 CATGGATCCC GCTCGAGGCG AGCGGGCTCC 30 42 base pairs nucleic aciddouble linear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001515-85.1 ~23.2 %G 111 CTGGTTCGGC CCAGAATTCT ATGGGTCTCG CGCGGCTCGT GG 4242 base pairs nucleic acid double linear DNA (genomic) NO NO Swinepoxvirus Kasza S-SPV-001 515-85.1 ~23.2 %G 112 CTCGCTCGCC CAGGATCCCTAGCGGAGGAT GGACTTGAGT CG 42 3628 base pairs nucleic acid double linearDNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G CDS57..1226 CDS 1362..3395 113 TTGAAGATGA ATGCATAGAG GAAGATGATG TCGANACGTCATTATTTAAT GTATAAATGG 60 ATAAATTGTA TGCGGCAATA TTCGGCGTTT TTATGACATCTAAAGATGAT GATTTTAATA 120 ACTTTATAGA AGTTGTAAAA TCTGTATTAA CAGATACATCANCTAATCAT ACAATATCGT 180 CGTCCAATAA TAATACATGG ATATATATAT TTCTAGCGATATTATTTGGT GTTATGGNAT 240 TATTAGTTTT TANTTTGTAT GTAGAAGTTC CTAAACCNACTTANATGGAG GAAGCAGATA 300 ACCNACTCGT TNTAAATAGT ATTAGTGCTA GAGCATTGGNGGCATTTTTT GTATCTAAAA 360 NTANTGATAT GGTCGNTGAA NTAGTTNCCC AAAAATNTCCNCCAAAGAAG ANATCACAAA 420 TAAAACGCAT AGATACACGA ATTCCTATTG ATCTTATTAATCAACAATTC GTTAAAAGAT 480 TTAAACTAGA AAATTATAAA AATGGAATTT TATCCGTTCTTATCAATAGT TTAGTCGAAA 540 ATAATTACTT TGAACAAGAT GGTAAACTTA ATAGCAGTGATATTGATGAA TTAGTGCTCA 600 CAGACATAGA GAAAAAGATT TTATCGTTGA TTCCTAGATGTTCTCCTCTT TATATAGATA 660 TCAGTGACGT TAAAGTTCTC GCATCTAGGT TAANNAAAAGTGCTAAATCA TTTACGTTTA 720 ATGATCATGA ATATATTATA CAATCTGATA AAATAGAGGAATTAATAAAT AGTTTATCTA 780 GAAACCATGA TATTATACTA GATGAAAAAA GTTCTATTAAAGACAGCATA TATATACTAT 840 CTGATGATCT TTTGAATATA CTTCGTGAAA GATTATTTAGATGTCCACAG GTTAAAGATA 900 ATACTATTTC TAGAACACGT CTATATGATT ATTTTACTAGAGTGTCAAAG AAAGAAGAAG 960 CGAAAATATA CGTTATATTG AAAGATTTAA AGATTGCTGATATACTCGGT ATCGAAACAG 1020 TAACGATAGG ATCATTTGTA TATACGAAAT ATAGCATGTTGATTAATTCA ATTTCGTCTA 1080 ATGTTGATAG ATATTCAAAA AGGTTCCATG ACTCTTTTTATGAAGATATT GCGGAATTTA 1140 TAAAGGATAA TGAAAAAATT AATGTATCCA GAGTTGTTGAATGCCTTATC GTACCTAATA 1200 TTAATATAGA GTTATTAACT GAATAAGTAT ATATAAATGATTGTTTTTAT AATGTTTGTT 1260 ATCGCATTTA GTTTTGCTGT ATGGTTATCA TATACATTTTTAAGGCCGTA TATGATAAAT 1320 GAAAATATAT AAGCACTTAT TTTTGTTAGT ATAATAACACAATGCCGTCG TATATGTATC 1380 CGAAGAACGC AAGAAAAGTA ATTTCAAAGA TTATATCATTACAACTTGAT ATTAAAAAAC 1440 TTCCTAAAAA ATATATAAAT ACCATGTTAG AATTTGGTCTACATGGAAAT CTACCAGCTT 1500 GTATGTATAA AGATGCCGTA TCATATGATA TAAATAATATAAGATTTTTA CCTTATAATT 1560 GTGTTATGGT TAAAGATTTA ATAAATGTTA TAAAATCATCATCTGTAATA GATACTAGAT 1620 TACATCAATC TGTATTAAAA CATCGTAGAG CGTTAATAGATTACGGCGAT CAAGACATTA 1680 TCACTTTAAT GATCATTAAT AAGTTACTAT CGATAGATGATATATCCTAT ATATTAGATA 1740 AAAAAATAAT TCATGTAACA AAAATATTAA AAATAGACCCTACAGTAGCC AATTCAAACA 1800 TGAAACTGAA TAAGATAGAG CTTGTAGATG TAATAACATCAATACCTAAG TCTTCCTATA 1860 CATATTTATA TAATAATATG ATCATTGATC TCGATACATTATTATATTTA TCCGATGCAT 1920 TCCACATACC CCCCACACAT ATATCATTAC GTTCACTTAGAGATATAAAC AGGATTATTG 1980 AATTGCTTAA AAAATATCCG AATAATAATA TTATTGATTATATATCCGAT AGCATAAAAT 2040 CAAATAGTTC ATTCATTCAC ATACTTCATA TGATAATATCAAATATGTTT CCTGCTATAA 2100 TCCCTAGTGT AAACGATTTT ATATCTACCG TAGTTGATAAAGATCGACTT ATTAATATGT 2160 ATGGGATTAA GTGTGTTGCT ATGTTTTCGT ACGATATAAACATGATCGAT TTAGAGTCAT 2220 TAGATGACTC AGATTACATA TTTATAGAAA AAAATATATCTATATACGAC GTTAAATGTA 2280 GAGATTTTGC GAATATGATT AGAGATAAGG TTAAAAGAGAAAAGAATAGA ATATTAACTA 2340 CGAAATGTGA AGATATTATA AGATATATAA AATTATTCAGTAAAAATAGA ATAAACGATG 2400 AAAATAATAA GGTGGAGGAG GTGTTGATAC ATATTGATAATGTATCTAAA AATAATAAAT 2460 TATCACTGTC TGATATATCA TCTTTAATGG ATCAATTTCGTTTAAATCCA TGTACCATAA 2520 GAAATATATT ATTATCTTCA GCAACTATAA AATCAAAACTATTAGCGTTA CGGGCAGTAA 2580 AAAACTGGAA ATGTTATTCA TTGACAAATG TATCAATGTATAAAAAAATA AAGGGTGTTA 2640 TCGTAATGGA TATGGTTGAT TATATATCTA CTAACATTCTTAAATACCAT AAACAATTAT 2700 ATGATAAAAT GAGTACGTTT GAATATAAAC GAGATATTAAATCATGTAAA TGCTCGATAT 2760 GTTCCGACTC TATAACACAT CATATATATG AAACAACATCATGTATAAAT TATAAATCTA 2820 CCGATAATGA TCTTATGATA GTATTGTTCA ATCTAACTAGATATTTAATG CATGGGATGA 2880 TACATCCTAA TCTTATAAGC GTAAAAGGAT GGGGTCCCCTTATTGGATTA TTAACGGGTG 2940 ATATAGGTAT TAATTTAAAA CTATATTCCA CCATGAATATAAATGGGCTA CGGTATGGAG 3000 ATATTACGTT ATCTTCATAC GATATGAGTA ATAAATTAGTCTCTATTATT AATACACCCA 3060 TATATGAGTT AATACCGTTT ACTACATGTT GTTCACTCAATGAATATTAT TCAAAAATTG 3120 TGATTTTAAT AAATGTTATT TTAGAATATA TGATATCTATTATATTATAT AGAATATTGA 3180 TCGTAAAAAG ATTTAATAAC ATTAAAGAAT TTATTTCAAAAGTCGTAAAT ACTGTACTAG 3240 AATCATCAGG CATATATTTT TGTCAGATGC GTGTACATGAACAAATTGAA TTGGAAATAG 3300 ATGAGCTCAT TATTAATGGA TCTATGCCTG TACAGCTTATGCATTTACTT CTAAAGGTAG 3360 CTACCATAAT ATTAGAGGAA ATCAAAGAAA TATAACGTATTTTTTCTTTT AAATAAATAA 3420 AAATACTTTT TTTTTTAAAC AAGGGGTGCT ACCTTGTCTAATTGTATCTT GTATTTTGGA 3480 TCTGATGCAA GATTATTAAA TAATCGTATG AAAAAGTAGTAGATATAGTT TATATCGTTA 3540 CTGGACATGA TATTATGTTT AGTTAATTCT TCTTTGGCATGAATTCTACA CGTCGGANAA 3600 GGTAATGTAT CTATAATGGT ATAAAGCT 3628 389 aminoacids amino acid double linear DNA (genomic) NO NO Swinepox virus KaszaS-SPV-001 515-85.1 ~23.2 %G 114 Met Asp Lys Leu Tyr Ala Ala Ile Phe GlyVal Phe Met Thr Ser Lys 1 5 10 15 Asp Asp Asp Phe Asn Asn Phe Ile GluVal Val Lys Ser Val Leu Thr 20 25 30 Asp Thr Ser Xaa Asn His Thr Ile SerSer Ser Asn Asn Asn Thr Trp 35 40 45 Ile Tyr Ile Phe Leu Ala Ile Leu PheGly Val Met Xaa Leu Leu Val 50 55 60 Phe Xaa Leu Tyr Val Glu Val Pro LysPro Thr Xaa Met Glu Glu Ala 65 70 75 80 Asp Asn Xaa Leu Val Xaa Asn SerIle Ser Ala Arg Ala Leu Xaa Ala 85 90 95 Phe Phe Val Ser Lys Xaa Xaa AspMet Val Xaa Glu Xaa Val Xaa Gln 100 105 110 Lys Xaa Pro Pro Lys Lys XaaSer Gln Ile Lys Arg Ile Asp Thr Arg 115 120 125 Ile Pro Ile Asp Leu IleAsn Gln Gln Phe Val Lys Arg Phe Lys Leu 130 135 140 Glu Asn Tyr Lys AsnGly Ile Leu Ser Val Leu Ile Asn Ser Leu Val 145 150 155 160 Glu Asn AsnTyr Phe Glu Gln Asp Gly Lys Leu Asn Ser Ser Asp Ile 165 170 175 Asp GluLeu Val Leu Thr Asp Ile Glu Lys Lys Ile Leu Ser Leu Ile 180 185 190 ProArg Cys Ser Pro Leu Tyr Ile Asp Ile Ser Asp Val Lys Val Leu 195 200 205Ala Ser Arg Leu Xaa Lys Ser Ala Lys Ser Phe Thr Phe Asn Asp His 210 215220 Glu Tyr Ile Ile Gln Ser Asp Lys Ile Glu Glu Leu Ile Asn Ser Leu 225230 235 240 Ser Arg Asn His Asp Ile Ile Leu Asp Glu Lys Ser Ser Ile LysAsp 245 250 255 Ser Ile Tyr Ile Leu Ser Asp Asp Leu Leu Asn Ile Leu ArgGlu Arg 260 265 270 Leu Phe Arg Cys Pro Gln Val Lys Asp Asn Thr Ile SerArg Thr Arg 275 280 285 Leu Tyr Asp Tyr Phe Thr Arg Val Ser Lys Lys GluGlu Ala Lys Ile 290 295 300 Tyr Val Ile Leu Lys Asp Leu Lys Ile Ala AspIle Leu Gly Ile Glu 305 310 315 320 Thr Val Thr Ile Gly Ser Phe Val TyrThr Lys Tyr Ser Met Leu Ile 325 330 335 Asn Ser Ile Ser Ser Asn Val AspArg Tyr Ser Lys Arg Phe His Asp 340 345 350 Ser Phe Tyr Glu Asp Ile AlaGlu Phe Ile Lys Asp Asn Glu Lys Ile 355 360 365 Asn Val Ser Arg Val ValGlu Cys Leu Ile Val Pro Asn Ile Asn Ile 370 375 380 Glu Leu Leu Thr Glu385 677 amino acids amino acid double linear DNA (genomic) NO NOSwinepox virus Kasza S-SPV-001 515-85.1 ~23.2 %G 115 Met Pro Ser Tyr MetTyr Pro Lys Asn Ala Arg Lys Val Ile Ser Lys 1 5 10 15 Ile Ile Ser LeuGln Leu Asp Ile Lys Lys Leu Pro Lys Lys Tyr Ile 20 25 30 Asn Thr Met LeuGlu Phe Gly Leu His Gly Asn Leu Pro Ala Cys Met 35 40 45 Tyr Lys Asp AlaVal Ser Tyr Asp Ile Asn Asn Ile Arg Phe Leu Pro 50 55 60 Tyr Asn Cys ValMet Val Lys Asp Leu Ile Asn Val Ile Lys Ser Ser 65 70 75 80 Ser Val IleAsp Thr Arg Leu His Gln Ser Val Leu Lys His Arg Arg 85 90 95 Ala Leu IleAsp Tyr Gly Asp Gln Asp Ile Ile Thr Leu Met Ile Ile 100 105 110 Asn LysLeu Leu Ser Ile Asp Asp Ile Ser Tyr Ile Leu Asp Lys Lys 115 120 125 IleIle His Val Thr Lys Ile Leu Lys Ile Asp Pro Thr Val Ala Asn 130 135 140Ser Asn Met Lys Leu Asn Lys Ile Glu Leu Val Asp Val Ile Thr Ser 145 150155 160 Ile Pro Lys Ser Ser Tyr Thr Tyr Leu Tyr Asn Asn Met Ile Ile Asp165 170 175 Leu Asp Thr Leu Leu Tyr Leu Ser Asp Ala Phe His Ile Pro ProThr 180 185 190 His Ile Ser Leu Arg Ser Leu Arg Asp Ile Asn Arg Ile IleGlu Leu 195 200 205 Leu Lys Lys Tyr Pro Asn Asn Asn Ile Ile Asp Tyr IleSer Asp Ser 210 215 220 Ile Lys Ser Asn Ser Ser Phe Ile His Ile Leu HisMet Ile Ile Ser 225 230 235 240 Asn Met Phe Pro Ala Ile Ile Pro Ser ValAsn Asp Phe Ile Ser Thr 245 250 255 Val Val Asp Lys Asp Arg Leu Ile AsnMet Tyr Gly Ile Lys Cys Val 260 265 270 Ala Met Phe Ser Tyr Asp Ile AsnMet Ile Asp Leu Glu Ser Leu Asp 275 280 285 Asp Ser Asp Tyr Ile Phe IleGlu Lys Asn Ile Ser Ile Tyr Asp Val 290 295 300 Lys Cys Arg Asp Phe AlaAsn Met Ile Arg Asp Lys Val Lys Arg Glu 305 310 315 320 Lys Asn Arg IleLeu Thr Thr Lys Cys Glu Asp Ile Ile Arg Tyr Ile 325 330 335 Lys Leu PheSer Lys Asn Arg Ile Asn Asp Glu Asn Asn Lys Val Glu 340 345 350 Glu ValLeu Ile His Ile Asp Asn Val Ser Lys Asn Asn Lys Leu Ser 355 360 365 LeuSer Asp Ile Ser Ser Leu Met Asp Gln Phe Arg Leu Asn Pro Cys 370 375 380Thr Ile Arg Asn Ile Leu Leu Ser Ser Ala Thr Ile Lys Ser Lys Leu 385 390395 400 Leu Ala Leu Arg Ala Val Lys Asn Trp Lys Cys Tyr Ser Leu Thr Asn405 410 415 Val Ser Met Tyr Lys Lys Ile Lys Gly Val Ile Val Met Asp MetVal 420 425 430 Asp Tyr Ile Ser Thr Asn Ile Leu Lys Tyr His Lys Gln LeuTyr Asp 435 440 445 Lys Met Ser Thr Phe Glu Tyr Lys Arg Asp Ile Lys SerCys Lys Cys 450 455 460 Ser Ile Cys Ser Asp Ser Ile Thr His His Ile TyrGlu Thr Thr Ser 465 470 475 480 Cys Ile Asn Tyr Lys Ser Thr Asp Asn AspLeu Met Ile Val Leu Phe 485 490 495 Asn Leu Thr Arg Tyr Leu Met His GlyMet Ile His Pro Asn Leu Ile 500 505 510 Ser Val Lys Gly Trp Gly Pro LeuIle Gly Leu Leu Thr Gly Asp Ile 515 520 525 Gly Ile Asn Leu Lys Leu TyrSer Thr Met Asn Ile Asn Gly Leu Arg 530 535 540 Tyr Gly Asp Ile Thr LeuSer Ser Tyr Asp Met Ser Asn Lys Leu Val 545 550 555 560 Ser Ile Ile AsnThr Pro Ile Tyr Glu Leu Ile Pro Phe Thr Thr Cys 565 570 575 Cys Ser LeuAsn Glu Tyr Tyr Ser Lys Ile Val Ile Leu Ile Asn Val 580 585 590 Ile LeuGlu Tyr Met Ile Ser Ile Ile Leu Tyr Arg Ile Leu Ile Val 595 600 605 LysArg Phe Asn Asn Ile Lys Glu Phe Ile Ser Lys Val Val Asn Thr 610 615 620Val Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gln Met Arg Val His Glu 625 630635 640 Gln Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile Asn Gly Ser Met Pro645 650 655 Val Gln Leu Met His Leu Leu Leu Lys Val Ala Thr Ile Ile LeuGlu 660 665 670 Glu Ile Lys Glu Ile 675 43 base pairs nucleic aciddouble linear DNA (genomic) NO NO Infectious bovine rhinotracheitisvirus Cooper Strain 116 CTGGTTCGGC CCAGAATTCG ATGCAACCCA CCGCGCCGCC CCG43 42 base pairs nucleic acid double linear DNA (genomic) NO NOInfectious bovine rhinotracheitis virus Cooper Strain 117 CTCGCTCGCCCAGGATCCCT AGCGGAGGAT GGACTTGAGT CG 42 31 base pairs nucleic acid doublelinear DNA (genomic) NO NO Equine Influenza A neuraminidase Prague/56118 GGGATCCATG AATCCTAATC AAAAACTCTT T 31 31 base pairs nucleic aciddouble linear DNA (genomic) NO NO Equine Influenza A neuraminidasePrague/56 119 GGGATCCTTA CGAAAAGTAT TTAATTTGTG C 31 42 base pairsnucleic acid double linear DNA (genomic) NO NO Equine influenza Ahemagglutinin 120 GGAGGCCTTC ATGACAGACA ACCATTATTT TGATACTACT GA 42 40base pairs nucleic acid double linear DNA (genomic) NO NO Equineinfluenza A hemagglutinin 121 GAAGGCCTTC TCAAATGCAA ATGTTGCATCTGATGTTGCC 40 32 base pairs nucleic acid double linear DNA (genomic) NONO Equine Influenza A hemagglutinin Prague/56 122 GGGATCCATG AACACTCAAATTCTAATATT AG 32 30 base pairs nucleic acid double linear DNA (genomic)NO NO Equine Influenza A hemagglutinin Prague/56 123 GGGATCCTTATATACAAATA GTGCACCGCA 30 30 base pairs nucleic acid double linear DNA(genomic) NO NO Equine Influenza A neuraminidase 124 GGGTCGACATGAATCCAAAT CAAAAGATAA 30 29 base pairs nucleic acid double linear DNA(genomic) NO NO Equine Influenza A neuraminidase 125 GGGTCGACTTACATCTTATC GATGTCAAA 29 33 base pairs nucleic acid double linear DNA(genomic) NO NO Human 126 CTCGAATTCG AAGTGGGCAA CGTGGATCCT CGC 33 24base pairs nucleic acid double linear DNA (genomic) NO NO Human 127CAGTTAGCCT CCCCCATCTC CCCA 24 21 base pairs nucleic acid double linearDNA (genomic) NO NO Equine herpesvirus type 1 128 CGGAATTCCT CTGGTTGCCGT 21 22 base pairs nucleic acid double linear DNA (genomic) NO NO Equineherpesvirus type 1 129 GACGGTGGAT CCGGTAGGCG GT 22 34 base pairs nucleicacid double linear DNA (genomic) NO NO Bovine parainfluenza-3 virus 130TTATGGATCC TGCTGCTGTG TTGAACAACT TTGT 34 38 base pairs nucleic aciddouble linear DNA (genomic) NO NO Bovine parainfluenza-3 virus 131CCGCGGATCC CATGACCATC ACAACCATAA TCATAGCC 38 43 base pairs nucleic aciddouble linear DNA (genomic) NO NO Bovine parainfluenza-3 virus 132CGTCGGATCC CTTAGCTGCA GTTTTTTGGA ACTTCTGTTT TGA 43 40 base pairs nucleicacid double linear DNA (genomic) NO NO Bovine parainfluenza-3 virus 133CATAGGATCC CATGGAATAT TGGAAACACA CAAACAGCAC 40 42 base pairs nucleicacid double linear DNA (genomic) NO NO Bovine viral diarrhea virusSinger Strain 134 ACGTCGGATC CCTTACCAAA CCACGTCTTA CTCTTGTTTT CC 42 40base pairs nucleic acid double linear DNA (genomic) NO NO Bovine viraldiarrhea virus Singer Strain 135 ACATAGGATC CCATGGGAGA AAACATAACACAGTGGAACC 40 33 base pairs nucleic acid double linear DNA (genomic) NONO Bovine viral diarrhea virus Singer Strain 136 CGTGGATCCT CAATTACAAGAGGTATCGTC TAC 33 31 base pairs nucleic acid double linear DNA (genomic)NO NO Bovine viral diarrhea virus Singer Strain 137 CATAGATCTTGTGGTGCTGT CCGACTTCGC A 31 37 base pairs nucleic acid double linear DNA(genomic) NO NO Bovine respiratory syncytial virus Strain 375 138TGCAGGATCC TCATTTACTA AAGGAAAGAT TGTTGAT 37 35 base pairs nucleic aciddouble linear DNA (genomic) NO NO Bovine respiratory syncytial virusStrain 375 139 CTCTGGATCC TACAGCCATG AGGATGATCA TCAGC 35 40 base pairsnucleic acid double linear DNA (genomic) NO NO Bovine respiratorysyncytial virus Strain 375 140 CGTCGGATCC CTCACAGTTC CACATCATTGTCTTTGGGAT 40 41 base pairs nucleic acid double linear DNA (genomic) NONO Bovine respiratory syncytial virus Strain 375 141 CTTAGGATCCCATGGCTCTT AGCAAGGTCA AACTAAATGA C 41 41 base pairs nucleic acid doublelinear DNA (genomic) NO NO Bovine respiratory syncytial virus Strain 375142 CGTTGGATCC CTAGATCTGT GTAGTTGATT GATTTGTGTG A 41 41 base pairsnucleic acid double linear DNA (genomic) NO NO Bovine respiratorysyncytial virus Strain 375 143 CTCTGGATCC TCATACCCAT CATCTTAAATTCAAGACATT A 41 51 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 144 ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCGCCCGGGGATC T 51 128 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 145 GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTTTTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAATAAATGAATT TGATCCATGA 120 ATCCTAAT 128 120 base pairs nucleic aciddouble linear DNA (genomic) NO NO not provided 146 CTTTTCGTAA GGATCAATTCGGATCCATAA TTAATTAATT TTTATCCCGG CGCGCCTCGA 60 CTCTAGAATT TCATTTTGTTTTTTTCTATG CTATAAATGA ATTCGGATCC CGTCGTTTTA 120 116 base pairs nucleicacid double linear DNA (genomic) NO NO not provided 147 GAAATCCAGCTGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA 60 TAATTAATTAACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 51 base pairsnucleic acid double linear DNA (genomic) NO NO not provided 148TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 149ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 168 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 150GTATTGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 60ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATT CACCCGCTGG 120TGGCGGTCTT TGGCGCGGGC CCCGTGGGCA TCGGCCCGGG CACCACGG 168 112 base pairsnucleic acid double linear DNA (genomic) NO NO not provided 151GAGCTCGAAT TCGGATCCAT AATTAATTAA TTTTTATCCC GGCGCGCCTC GACTCTAGAA 60TTTCATTTTG TTTTTTTCTA TGCTATAAAT GAATTCGGAT CCCGTCGTTT TA 112 116 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 152GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA 60TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 51base pairs nucleic acid double linear DNA (genomic) NO NO not provided153 TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 154ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 104 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 155AAATATATAA ATACCATGTT AGAATTTGGT CTGCTGCAGG TCGACTCTAG AATTTCATTT 60TGTTTTTTTC TATGCTATAA ATGAATTCGG ATCCCGTCGT TTTA 104 185 base pairsnucleic acid double linear DNA (genomic) NO NO not provided 156GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA 60TAATTAATTA ACCCGGTCGA CTCTAGAAAA AATTGAAAAA CTATTCTAAT TTATTGCACG 120GAGATCTTTT TTTTTTTTTT TTTTTTGGCA TATAAATGAA TTCGGATCCC CGGTGGCTTT 180GGGGG 185 66 base pairs nucleic acid double linear DNA (genomic) NO NOnot provided 157 CTCAATGTTA GGGTACCGAG CTCGAATTGG GTCGACCGGG TCGACCTGCAGCCTACATGG 60 AAATCT 66 51 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 158 TAATGTATCT ATAATGGTAT AAAGCTTGTATTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 159 ACAGGAAACA GCTATGACCA TGATTACGAATTCGAGCTCG CCCGGGGATC T 51 127 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 160 GTATAGCGGC CGCCTGCAGG TCGACTCTAGATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAAT TGAATTGTAATTATCGATAA TAAATGAATT TCGACATGAA 120 TCCAAAT 127 122 base pairs nucleicacid double linear DNA (genomic) NO NO not provided 161 GATAAGATGTAAGTCGAAAT TCGGATCCAT AATTAATTAA TTTTTATCCC GGCGCGCCTC 60 GACTCTAGAATTTCATTTTG TTTTTTTCTA TGCTATAAAT GAATTCGGAT CCCGTCGTTT 120 TA 122 116base pairs nucleic acid double linear DNA (genomic) NO NO not provided162 GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA AAAAGATCCA 60TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCG CTATAC 116 51base pairs nucleic acid double linear DNA (genomic) NO NO not provided163 TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 164ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 61 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 165GTATAGCGGC CGCCTGCAGG TCGACCTGCA GTGAATAATA AAATGTGTGT TTGTCCGAAA 60 T61 45 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 166 CTCCATAGAA GACACCGGGA CCATGGATCC CGTCGTTTTA CAACG 45 105base pairs nucleic acid double linear DNA (genomic) NO NO not provided167 TCGGCGGAAA TCCAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA 60GATCTAGAAT AAGCTAGAGG ATCGATCCCC TATGGCGATC ATCAG 105 31 base pairsnucleic acid double linear DNA (genomic) NO NO not provided 168CTGCAGGTCG ACCTGCAGGC GGCCGCTATA C 31 51 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 169 TAATGTATCT ATAATGGTATAAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 170 ACAGGAAACA GCTATGACCATGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 193 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 171 GTATAGCGGC CGCCTGCAGGTCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAATTGAATTGTAA TTATCGATAA TAAATGAATT CCGAAGTGGG 120 CAACGTGGAT CCTCGCCCTCGGGCTCCTCG TGGTCCGCAC CGTCGTGGCC AGAAGTGCTC 180 CTACTAGCTC GAG 193 123base pairs nucleic acid double linear DNA (genomic) NO NO not provided172 ATCATTAGCA CGTTAACTTA ATAAGATCCA TAATTAATTA ATTTTTATCC CGGCGCGCCT 60CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA TGAATTCGGA TCCCGTCGTT 120TTA 123 116 base pairs nucleic acid double linear DNA (genomic) NO NOnot provided 173 GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCAAAAAGATCCA 60 TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCGCTATAC 116 51 base pairs nucleic acid double linear DNA (genomic) NO NOnot provided 174 TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAATC 51 51 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 175 ACAGGAAACA GCTATGACCA TGATTACGAA TTCGAGCTCG CCCGGGGATC T 51133 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 176 GTATAGCGGC CGCCTGCAGG TCGACTCTAG ATTTTTTTTT TTTTTTTTTTTGGCATATAA 60 ATAGATCTGT ATCCTAAAAT TGAATTGTAA TTATCGATAA TAAATGAATTCCTCTGGTTG 120 CCGTTCTGTC GGC 133 99 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 177 GAAAATGAAA AAATGGTTTAAACCGGGGGC GCGCCTCGAC TCTAGAATTT CATTTTGTTT 60 TTTTCTATGC TATAAATGAATTCGGATCCC GTCGTTTTA 99 116 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 178 GAAATCCAGC TGAGCGCCGG TCGCTACCATTACCAGTTGG TCTGGTGTCA AAAAGATCCA 60 TAATTAATTA ACCCGGGTCG AGGCGCGCCGGGTCGACCTG CAGGCGGCCG CTATAC 116 51 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 179 TAATGTATCT ATAATGGTATAAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 180 ACAGGAAACA GCTATGACCATGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 140 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 181 GTATAGCGGC CGCCTGCAGGTCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAATTGAATTGTAA TTATCGATAA TAAATGAATT CGGATCAGCT 120 TATGATGGAT GGACGTTTGG140 123 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 182 GGAGGTGTCC ACGGCCTTAA AGCTGATCCA TAATTAATTA ATTTTTATCCCGGCGCGCCT 60 CGACTCTAGA ATTTCATTTT GTTTTTTTCT ATGCTATAAA TGAATTCGGATCCCGTCGTT 120 TTA 123 116 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 183 GAAATCCAGC TGAGCGCCGG TCGCTACCATTACCAGTTGG TCTGGTGTCA AAAAGATCCA 60 TAATTAATTA ACCCGGGTCG AGGCGCGCCGGGTCGACCTG CAGGCGGCCG CTATAC 116 51 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 184 TAATGTATCT ATAATGGTATAAAGCTTGTA TTCTATAGTG TCACCTAAAT C 51 39 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 185 GAAGCATGCC CGTTCTTATCAATAGTTTAG TCGAAAATA 39 41 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 186 CATAAGATCT GGCATTGTGT TATTATACTAACAAAAATAA G 41 41 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 187 CCGTAGTCGA CAAAGATCGA CTTATTAATA TGTATGGGAT T 4139 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 188 GCCTGAAGCT TCTAGTACAG TATTTACGAC TTTTGAAAT 39 3942 basepairs nucleic acid double linear DNA (genomic) NO NO Swinepox virusKasza S-SPV-001 CDS 1..369 CDS 370..597 CDS 598..1539 CDS 1675..3708 CDScomplement (3748..3942) 189 TGT TTG TTC ATT AAT AAG ATG GGT GGA GCT ATTATA GAA TAC AAG ATA 48 Cys Leu Phe Ile Asn Lys Met Gly Gly Ala Ile IleGlu Tyr Lys Ile 1 5 10 15 CCT GGT TCC AAA TCT ATA ACC AAA TCT ATT TCCGAA GAA CTA GAA AAT 96 Pro Gly Ser Lys Ser Ile Thr Lys Ser Ile Ser GluGlu Leu Glu Asn 20 25 30 TTA ACA AAG CGA GAT AAA CCA ATA TCT AAA ATT ATAGTT ATT CCT ATT 144 Leu Thr Lys Arg Asp Lys Pro Ile Ser Lys Ile Ile ValIle Pro Ile 35 40 45 GTA TGT TAC AGA AAT GCA AAT AGT ATA AAG GTT ACA TTTGCA CTA AAA 192 Val Cys Tyr Arg Asn Ala Asn Ser Ile Lys Val Thr Phe AlaLeu Lys 50 55 60 AAG TTT ATC ATA GAT AAG GAG TTT AGT ACA AAT GTA ATA GACGTA GAT 240 Lys Phe Ile Ile Asp Lys Glu Phe Ser Thr Asn Val Ile Asp ValAsp 65 70 75 80 GGT AAA CAT GAA AAA ATG TCC ATG AAT GAA ACA TGC GAA GAGGAT GTT 288 Gly Lys His Glu Lys Met Ser Met Asn Glu Thr Cys Glu Glu AspVal 85 90 95 GCT AGA GGA TTG GGA ATT ATA GAT CTT GAA GAT GAA TGC ATA GAGGAA 336 Ala Arg Gly Leu Gly Ile Ile Asp Leu Glu Asp Glu Cys Ile Glu Glu100 105 110 GAT GAT GTC GAT ACG TCA TTA TTT AAT GTA TAAATG GAT AAA TTGTAT 384 Asp Asp Val Asp Thr Ser Leu Phe Asn Val Met Asp Lys Leu Tyr 115120 1 5 GCG GCA ATA TTC GGC GTT TTT ATG ACA TCT AAA GAT GAT GAT TTT AAT432 Ala Ala Ile Phe Gly Val Phe Met Thr Ser Lys Asp Asp Asp Phe Asn 1015 20 AAC TTT ATA GAA GTT GTA AAA TCT GTA TTA ACA GAT ACA TCA TCT AAT480 Asn Phe Ile Glu Val Val Lys Ser Val Leu Thr Asp Thr Ser Ser Asn 2530 35 CAT ACA ATA TCG TCG TCC AAT AAT AAT ACA TGG ATA TAT ATA TTT CTA528 His Thr Ile Ser Ser Ser Asn Asn Asn Thr Trp Ile Tyr Ile Phe Leu 4045 50 GCG ATA TTA TTT GGT GTT ATG GTA TTA TTA GTT TTT ATT TTG TAT TTA576 Ala Ile Leu Phe Gly Val Met Val Leu Leu Val Phe Ile Leu Tyr Leu 5560 65 AAA GTT ACT AAA CCA ACT TAAATG GAG GAA GCA GAT AAC CAA CTC GTT 624Lys Val Thr Lys Pro Thr Met Glu Glu Ala Asp Asn Gln Leu Val 70 75 1 5TTA AAT AGT ATT AGT GCT AGA GCA TTA AAG GCA TTT TTT GTA TCT AAA 672 LeuAsn Ser Ile Ser Ala Arg Ala Leu Lys Ala Phe Phe Val Ser Lys 10 15 20 25ATT AAT GAT ATG GTC GAT GAA TTA GTT ACC AAA AAA TAT CCA CCA AAG 720 IleAsn Asp Met Val Asp Glu Leu Val Thr Lys Lys Tyr Pro Pro Lys 30 35 40 AAGAAA TCA CAA ATA AAA CTC ATA GAT ACA CGA ATT CCT ATT GAT CTT 768 Lys LysSer Gln Ile Lys Leu Ile Asp Thr Arg Ile Pro Ile Asp Leu 45 50 55 ATT AATCAA CAA TTC GTT AAA AGA TTT AAA CTA GAA AAT TAT AAA AAT 816 Ile Asn GlnGln Phe Val Lys Arg Phe Lys Leu Glu Asn Tyr Lys Asn 60 65 70 GGA ATT TTATCC GTT CTT ATC AAT AGT TTA GTC GAA AAT AAT TAC TTT 864 Gly Ile Leu SerVal Leu Ile Asn Ser Leu Val Glu Asn Asn Tyr Phe 75 80 85 GAA CAA GAT GGTAAA CTT AAT AGC AGT GAT ATT GAT GAA TTA GTG CTC 912 Glu Gln Asp Gly LysLeu Asn Ser Ser Asp Ile Asp Glu Leu Val Leu 90 95 100 105 ACA GAC ATAGAG AAA AAG ATT TTA TCG TTG ATT CCT AGA TGT TCT CCT 960 Thr Asp Ile GluLys Lys Ile Leu Ser Leu Ile Pro Arg Cys Ser Pro 110 115 120 CTT TAT ATAGAT ATC AGT GAC GTT AAA GTT CTC GCA TCT AGG TTA AAA 1008 Leu Tyr Ile AspIle Ser Asp Val Lys Val Leu Ala Ser Arg Leu Lys 125 130 135 AAA AGT GCTAAA TCA TTT ACG TTT AAT GAT CAT GAA TAT ATT ATA CAA 1056 Lys Ser Ala LysSer Phe Thr Phe Asn Asp His Glu Tyr Ile Ile Gln 140 145 150 TCT GAT AAAATA GAG GAA TTA ATA AAT AGT TTA TCT AGA AAC CAT GAT 1104 Ser Asp Lys IleGlu Glu Leu Ile Asn Ser Leu Ser Arg Asn His Asp 155 160 165 ATT ATA CTAGAT GAA AAA AGT TCT ATT AAA GAC AGC ATA TAT ATA CTA 1152 Ile Ile Leu AspGlu Lys Ser Ser Ile Lys Asp Ser Ile Tyr Ile Leu 170 175 180 185 TCT GATGAT CTT TTG AAT ATA CTT CGT GAA AGA TTA TTT AGA TGT CCA 1200 Ser Asp AspLeu Leu Asn Ile Leu Arg Glu Arg Leu Phe Arg Cys Pro 190 195 200 CAG GTTAAA GAT AAT ACT ATT TCT AGA ACA CGT CTA TAT GAT TAT TTT 1248 Gln Val LysAsp Asn Thr Ile Ser Arg Thr Arg Leu Tyr Asp Tyr Phe 205 210 215 ACT AGAGTG TCA AAG AAA GAA GAA GCG AAA ATA TAC GTT ATA TTG AAA 1296 Thr Arg ValSer Lys Lys Glu Glu Ala Lys Ile Tyr Val Ile Leu Lys 220 225 230 GAT TTAAAG ATT GCT GAT ATA CTC GGT ATC GAA ACA GTA ACG ATA GGA 1344 Asp Leu LysIle Ala Asp Ile Leu Gly Ile Glu Thr Val Thr Ile Gly 235 240 245 TCA TTTGTA TAT ACG AAA TAT AGC ATG TTG ATT AAT TCA ATT TCG TCT 1392 Ser Phe ValTyr Thr Lys Tyr Ser Met Leu Ile Asn Ser Ile Ser Ser 250 255 260 265 AATGTT GAT AGA TAT TCA AAA AGG TTC CAT GAC TCT TTT TAT GAA GAT 1440 Asn ValAsp Arg Tyr Ser Lys Arg Phe His Asp Ser Phe Tyr Glu Asp 270 275 280 ATTGCG GAA TTT ATA AAG GAT AAT GAA AAA ATT AAT GTA TCC AGA GTT 1488 Ile AlaGlu Phe Ile Lys Asp Asn Glu Lys Ile Asn Val Ser Arg Val 285 290 295 GTTGAA TGC CTT ATC GTA CCT AAT ATT AAT ATA GAG TTA TTA ACT GAA 1536 Val GluCys Leu Ile Val Pro Asn Ile Asn Ile Glu Leu Leu Thr Glu 300 305 310TAAGTATATA TAAATGATTG TTTTTATAAT GTTTGTTATC GCATTTAGTT TTGCTGTATG 1596GTTATCATAT ACATTTTTAA GGCCGTATAT GATAAATGAA AATATATAAG CACTTATTTT 1656TGTTAGTATA ATAACACA ATG CCG TCG TAT ATG TAT CCG AAG AAC GCA AGA 1707 MetPro Ser Tyr Met Tyr Pro Lys Asn Ala Arg 1 5 10 AAA GTA ATT TCA AAG ATTATA TCA TTA CAA CTT GAT ATT AAA AAA CTT 1755 Lys Val Ile Ser Lys Ile IleSer Leu Gln Leu Asp Ile Lys Lys Leu 15 20 25 CCT AAA AAA TAT ATA AAT ACCATG TTA GAA TTT GGT CTA CAT GGA AAT 1803 Pro Lys Lys Tyr Ile Asn Thr MetLeu Glu Phe Gly Leu His Gly Asn 30 35 40 CTA CCA GCT TGT ATG TAT AAA GATGCC GTA TCA TAT GAT ATA AAT AAT 1851 Leu Pro Ala Cys Met Tyr Lys Asp AlaVal Ser Tyr Asp Ile Asn Asn 45 50 55 ATA AGA TTT TTA CCT TAT AAT TGT GTTATG GTT AAA GAT TTA ATA AAT 1899 Ile Arg Phe Leu Pro Tyr Asn Cys Val MetVal Lys Asp Leu Ile Asn 60 65 70 75 GTT ATA AAA TCA TCA TCT GTA ATA GATACT AGA TTA CAT CAA TCT GTA 1947 Val Ile Lys Ser Ser Ser Val Ile Asp ThrArg Leu His Gln Ser Val 80 85 90 TTA AAA CAT CGT AGA GCG TTA ATA GAT TACGGC GAT CAA GAC ATT ATC 1995 Leu Lys His Arg Arg Ala Leu Ile Asp Tyr GlyAsp Gln Asp Ile Ile 95 100 105 ACT TTA ATG ATC ATT AAT AAG TTA CTA TCGATA GAT GAT ATA TCC TAT 2043 Thr Leu Met Ile Ile Asn Lys Leu Leu Ser IleAsp Asp Ile Ser Tyr 110 115 120 ATA TTA GAT AAA AAA ATA ATT CAT GTA ACAAAA ATA TTA AAA ATA GAC 2091 Ile Leu Asp Lys Lys Ile Ile His Val Thr LysIle Leu Lys Ile Asp 125 130 135 CCT ACA GTA GCC AAT TCA AAC ATG AAA CTGAAT AAG ATA GAG CTT GTA 2139 Pro Thr Val Ala Asn Ser Asn Met Lys Leu AsnLys Ile Glu Leu Val 140 145 150 155 GAT GTA ATA ACA TCA ATA CCT AAG TCTTCC TAT ACA TAT TTA TAT AAT 2187 Asp Val Ile Thr Ser Ile Pro Lys Ser SerTyr Thr Tyr Leu Tyr Asn 160 165 170 AAT ATG ATC ATT GAT CTC GAT ACA TTATTA TAT TTA TCC GAT GCA TTC 2235 Asn Met Ile Ile Asp Leu Asp Thr Leu LeuTyr Leu Ser Asp Ala Phe 175 180 185 CAC ATA CCC CCC ACA CAT ATA TCA TTACGT TCA CTT AGA GAT ATA AAC 2283 His Ile Pro Pro Thr His Ile Ser Leu ArgSer Leu Arg Asp Ile Asn 190 195 200 AGG ATT ATT GAA TTG CTT AAA AAA TATCCG AAT AAT AAT ATT ATT GAT 2331 Arg Ile Ile Glu Leu Leu Lys Lys Tyr ProAsn Asn Asn Ile Ile Asp 205 210 215 TAT ATA TCC GAT AGC ATA AAA TCA AATAGT TCA TTC ATT CAC ATA CTT 2379 Tyr Ile Ser Asp Ser Ile Lys Ser Asn SerSer Phe Ile His Ile Leu 220 225 230 235 CAT ATG ATA ATA TCA AAT ATG TTTCCT GCT ATA ATC CCT AGT GTA AAC 2427 His Met Ile Ile Ser Asn Met Phe ProAla Ile Ile Pro Ser Val Asn 240 245 250 GAT TTT ATA TCT ACC GTA GTT GATAAA GAT CGA CTT ATT AAT ATG TAT 2475 Asp Phe Ile Ser Thr Val Val Asp LysAsp Arg Leu Ile Asn Met Tyr 255 260 265 GGG ATT AAG TGT GTT GCT ATG TTTTCG TAC GAT ATA AAC ATG ATC GAT 2523 Gly Ile Lys Cys Val Ala Met Phe SerTyr Asp Ile Asn Met Ile Asp 270 275 280 TTA GAG TCA TTA GAT GAC TCA GATTAC ATA TTT ATA GAA AAA AAT ATA 2571 Leu Glu Ser Leu Asp Asp Ser Asp TyrIle Phe Ile Glu Lys Asn Ile 285 290 295 TCT ATA TAC GAC GTT AAA TGT AGAGAT TTT GCG AAT ATG ATT AGA GAT 2619 Ser Ile Tyr Asp Val Lys Cys Arg AspPhe Ala Asn Met Ile Arg Asp 300 305 310 315 AAG GTT AAA AGA GAA AAG AATAGA ATA TTA ACT ACG AAA TGT GAA GAT 2667 Lys Val Lys Arg Glu Lys Asn ArgIle Leu Thr Thr Lys Cys Glu Asp 320 325 330 ATT ATA AGA TAT ATA AAA TTATTC AGT AAA AAT AGA ATA AAC GAT GAA 2715 Ile Ile Arg Tyr Ile Lys Leu PheSer Lys Asn Arg Ile Asn Asp Glu 335 340 345 AAT AAT AAG GTG GAG GAG GTGTTG ATA CAT ATT GAT AAT GTA TCT AAA 2763 Asn Asn Lys Val Glu Glu Val LeuIle His Ile Asp Asn Val Ser Lys 350 355 360 AAT AAT AAA TTA TCA CTG TCTGAT ATA TCA TCT TTA ATG GAT CAA TTT 2811 Asn Asn Lys Leu Ser Leu Ser AspIle Ser Ser Leu Met Asp Gln Phe 365 370 375 CGT TTA AAT CCA TGT ACC ATAAGA AAT ATA TTA TTA TCT TCA GCA ACT 2859 Arg Leu Asn Pro Cys Thr Ile ArgAsn Ile Leu Leu Ser Ser Ala Thr 380 385 390 395 ATA AAA TCA AAA CTA TTAGCG TTA CGG GCA GTA AAA AAC TGG AAA TGT 2907 Ile Lys Ser Lys Leu Leu AlaLeu Arg Ala Val Lys Asn Trp Lys Cys 400 405 410 TAT TCA TTG ACA AAT GTATCA ATG TAT AAA AAA ATA AAG GGT GTT ATC 2955 Tyr Ser Leu Thr Asn Val SerMet Tyr Lys Lys Ile Lys Gly Val Ile 415 420 425 GTA ATG GAT ATG GTT GATTAT ATA TCT ACT AAC ATT CTT AAA TAC CAT 3003 Val Met Asp Met Val Asp TyrIle Ser Thr Asn Ile Leu Lys Tyr His 430 435 440 AAA CAA TTA TAT GAT AAAATG AGT ACG TTT GAA TAT AAA CGA GAT ATT 3051 Lys Gln Leu Tyr Asp Lys MetSer Thr Phe Glu Tyr Lys Arg Asp Ile 445 450 455 AAA TCA TGT AAA TGC TCGATA TGT TCC GAC TCT ATA ACA CAT CAT ATA 3099 Lys Ser Cys Lys Cys Ser IleCys Ser Asp Ser Ile Thr His His Ile 460 465 470 475 TAT GAA ACA ACA TCATGT ATA AAT TAT AAA TCT ACC GAT AAT GAT CTT 3147 Tyr Glu Thr Thr Ser CysIle Asn Tyr Lys Ser Thr Asp Asn Asp Leu 480 485 490 ATG ATA GTA TTG TTCAAT CTA ACT AGA TAT TTA ATG CAT GGG ATG ATA 3195 Met Ile Val Leu Phe AsnLeu Thr Arg Tyr Leu Met His Gly Met Ile 495 500 505 CAT CCT AAT CTT ATAAGC GTA AAA GGA TGG GGT CCC CTT ATT GGA TTA 3243 His Pro Asn Leu Ile SerVal Lys Gly Trp Gly Pro Leu Ile Gly Leu 510 515 520 TTA ACG GGT GAT ATAGGT ATT AAT TTA AAA CTA TAT TCC ACC ATG AAT 3291 Leu Thr Gly Asp Ile GlyIle Asn Leu Lys Leu Tyr Ser Thr Met Asn 525 530 535 ATA AAT GGG CTA CGGTAT GGA GAT ATT ACG TTA TCT TCA TAC GAT ATG 3339 Ile Asn Gly Leu Arg TyrGly Asp Ile Thr Leu Ser Ser Tyr Asp Met 540 545 550 555 AGT AAT AAA TTAGTC TCT ATT ATT AAT ACA CCC ATA TAT GAG TTA ATA 3387 Ser Asn Lys Leu ValSer Ile Ile Asn Thr Pro Ile Tyr Glu Leu Ile 560 565 570 CCG TTT ACT ACATGT TGT TCA CTC AAT GAA TAT TAT TCA AAA ATT GTG 3435 Pro Phe Thr Thr CysCys Ser Leu Asn Glu Tyr Tyr Ser Lys Ile Val 575 580 585 ATT TTA ATA AATGTT ATT TTA GAA TAT ATG ATA TCT ATT ATA TTA TAT 3483 Ile Leu Ile Asn ValIle Leu Glu Tyr Met Ile Ser Ile Ile Leu Tyr 590 595 600 AGA ATA TTG ATCGTA AAA AGA TTT AAT AAC ATT AAA GAA TTT ATT TCA 3531 Arg Ile Leu Ile ValLys Arg Phe Asn Asn Ile Lys Glu Phe Ile Ser 605 610 615 AAA GTC GTA AATACT GTA CTA GAA TCA TCA GGC ATA TAT TTT TGT CAG 3579 Lys Val Val Asn ThrVal Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gln 620 625 630 635 ATG CGT GTACAT GAA CAA ATT GAA TTG GAA ATA GAT GAG CTC ATT ATT 3627 Met Arg Val HisGlu Gln Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile 640 645 650 AAT GGA TCTATG CCT GTA CAG CTT ATG CAT TTA CTT CTA AAG GTA GCT 3675 Asn Gly Ser MetPro Val Gln Leu Met His Leu Leu Leu Lys Val Ala 655 660 665 ACC ATA ATATTA GAG GAA ATC AAA GAA ATA TAACGTATTT TTTCTTTTAA 3725 Thr Ile Ile LeuGlu Glu Ile Lys Glu Ile 670 675 ATAAATAAAA ATACTTTTTT TTTTAAACAAGGGGTGCTAC CTTGTCTAAT TGTATCTTGT 3785 ATTTTGGATC TGATGCAAGA TTATTAAATAATCGTATGAA AAAGTAGTAG ATATAGTTTA 3845 TATCGTTACT GGACATGATA TTATGTTTAGTTAATTCTTC TTTGGCATGA ATTCTACACG 3905 TCGGACAAGG TAATGTATCT ATAATGGTATAAAGCTT 3942 122 amino acids amino acid linear peptide not provided 190Cys Leu Phe Ile Asn Lys Met Gly Gly Ala Ile Ile Glu Tyr Lys Ile 1 5 1015 Pro Gly Ser Lys Ser Ile Thr Lys Ser Ile Ser Glu Glu Leu Glu Asn 20 2530 Leu Thr Lys Arg Asp Lys Pro Ile Ser Lys Ile Ile Val Ile Pro Ile 35 4045 Val Cys Tyr Arg Asn Ala Asn Ser Ile Lys Val Thr Phe Ala Leu Lys 50 5560 Lys Phe Ile Ile Asp Lys Glu Phe Ser Thr Asn Val Ile Asp Val Asp 65 7075 80 Gly Lys His Glu Lys Met Ser Met Asn Glu Thr Cys Glu Glu Asp Val 8590 95 Ala Arg Gly Leu Gly Ile Ile Asp Leu Glu Asp Glu Cys Ile Glu Glu100 105 110 Asp Asp Val Asp Thr Ser Leu Phe Asn Val 115 120 75 aminoacids amino acid linear protein not provided 191 Met Asp Lys Leu Tyr AlaAla Ile Phe Gly Val Phe Met Thr Ser Lys 1 5 10 15 Asp Asp Asp Phe AsnAsn Phe Ile Glu Val Val Lys Ser Val Leu Thr 20 25 30 Asp Thr Ser Ser AsnHis Thr Ile Ser Ser Ser Asn Asn Asn Thr Trp 35 40 45 Ile Tyr Ile Phe LeuAla Ile Leu Phe Gly Val Met Val Leu Leu Val 50 55 60 Phe Ile Leu Tyr LeuLys Val Thr Lys Pro Thr 65 70 75 313 amino acids amino acid linearprotein not provided 192 Met Glu Glu Ala Asp Asn Gln Leu Val Leu Asn SerIle Ser Ala Arg 1 5 10 15 Ala Leu Lys Ala Phe Phe Val Ser Lys Ile AsnAsp Met Val Asp Glu 20 25 30 Leu Val Thr Lys Lys Tyr Pro Pro Lys Lys LysSer Gln Ile Lys Leu 35 40 45 Ile Asp Thr Arg Ile Pro Ile Asp Leu Ile AsnGln Gln Phe Val Lys 50 55 60 Arg Phe Lys Leu Glu Asn Tyr Lys Asn Gly IleLeu Ser Val Leu Ile 65 70 75 80 Asn Ser Leu Val Glu Asn Asn Tyr Phe GluGln Asp Gly Lys Leu Asn 85 90 95 Ser Ser Asp Ile Asp Glu Leu Val Leu ThrAsp Ile Glu Lys Lys Ile 100 105 110 Leu Ser Leu Ile Pro Arg Cys Ser ProLeu Tyr Ile Asp Ile Ser Asp 115 120 125 Val Lys Val Leu Ala Ser Arg LeuLys Lys Ser Ala Lys Ser Phe Thr 130 135 140 Phe Asn Asp His Glu Tyr IleIle Gln Ser Asp Lys Ile Glu Glu Leu 145 150 155 160 Ile Asn Ser Leu SerArg Asn His Asp Ile Ile Leu Asp Glu Lys Ser 165 170 175 Ser Ile Lys AspSer Ile Tyr Ile Leu Ser Asp Asp Leu Leu Asn Ile 180 185 190 Leu Arg GluArg Leu Phe Arg Cys Pro Gln Val Lys Asp Asn Thr Ile 195 200 205 Ser ArgThr Arg Leu Tyr Asp Tyr Phe Thr Arg Val Ser Lys Lys Glu 210 215 220 GluAla Lys Ile Tyr Val Ile Leu Lys Asp Leu Lys Ile Ala Asp Ile 225 230 235240 Leu Gly Ile Glu Thr Val Thr Ile Gly Ser Phe Val Tyr Thr Lys Tyr 245250 255 Ser Met Leu Ile Asn Ser Ile Ser Ser Asn Val Asp Arg Tyr Ser Lys260 265 270 Arg Phe His Asp Ser Phe Tyr Glu Asp Ile Ala Glu Phe Ile LysAsp 275 280 285 Asn Glu Lys Ile Asn Val Ser Arg Val Val Glu Cys Leu IleVal Pro 290 295 300 Asn Ile Asn Ile Glu Leu Leu Thr Glu 305 310 677amino acids amino acid linear protein not provided 193 Met Pro Ser TyrMet Tyr Pro Lys Asn Ala Arg Lys Val Ile Ser Lys 1 5 10 15 Ile Ile SerLeu Gln Leu Asp Ile Lys Lys Leu Pro Lys Lys Tyr Ile 20 25 30 Asn Thr MetLeu Glu Phe Gly Leu His Gly Asn Leu Pro Ala Cys Met 35 40 45 Tyr Lys AspAla Val Ser Tyr Asp Ile Asn Asn Ile Arg Phe Leu Pro 50 55 60 Tyr Asn CysVal Met Val Lys Asp Leu Ile Asn Val Ile Lys Ser Ser 65 70 75 80 Ser ValIle Asp Thr Arg Leu His Gln Ser Val Leu Lys His Arg Arg 85 90 95 Ala LeuIle Asp Tyr Gly Asp Gln Asp Ile Ile Thr Leu Met Ile Ile 100 105 110 AsnLys Leu Leu Ser Ile Asp Asp Ile Ser Tyr Ile Leu Asp Lys Lys 115 120 125Ile Ile His Val Thr Lys Ile Leu Lys Ile Asp Pro Thr Val Ala Asn 130 135140 Ser Asn Met Lys Leu Asn Lys Ile Glu Leu Val Asp Val Ile Thr Ser 145150 155 160 Ile Pro Lys Ser Ser Tyr Thr Tyr Leu Tyr Asn Asn Met Ile IleAsp 165 170 175 Leu Asp Thr Leu Leu Tyr Leu Ser Asp Ala Phe His Ile ProPro Thr 180 185 190 His Ile Ser Leu Arg Ser Leu Arg Asp Ile Asn Arg IleIle Glu Leu 195 200 205 Leu Lys Lys Tyr Pro Asn Asn Asn Ile Ile Asp TyrIle Ser Asp Ser 210 215 220 Ile Lys Ser Asn Ser Ser Phe Ile His Ile LeuHis Met Ile Ile Ser 225 230 235 240 Asn Met Phe Pro Ala Ile Ile Pro SerVal Asn Asp Phe Ile Ser Thr 245 250 255 Val Val Asp Lys Asp Arg Leu IleAsn Met Tyr Gly Ile Lys Cys Val 260 265 270 Ala Met Phe Ser Tyr Asp IleAsn Met Ile Asp Leu Glu Ser Leu Asp 275 280 285 Asp Ser Asp Tyr Ile PheIle Glu Lys Asn Ile Ser Ile Tyr Asp Val 290 295 300 Lys Cys Arg Asp PheAla Asn Met Ile Arg Asp Lys Val Lys Arg Glu 305 310 315 320 Lys Asn ArgIle Leu Thr Thr Lys Cys Glu Asp Ile Ile Arg Tyr Ile 325 330 335 Lys LeuPhe Ser Lys Asn Arg Ile Asn Asp Glu Asn Asn Lys Val Glu 340 345 350 GluVal Leu Ile His Ile Asp Asn Val Ser Lys Asn Asn Lys Leu Ser 355 360 365Leu Ser Asp Ile Ser Ser Leu Met Asp Gln Phe Arg Leu Asn Pro Cys 370 375380 Thr Ile Arg Asn Ile Leu Leu Ser Ser Ala Thr Ile Lys Ser Lys Leu 385390 395 400 Leu Ala Leu Arg Ala Val Lys Asn Trp Lys Cys Tyr Ser Leu ThrAsn 405 410 415 Val Ser Met Tyr Lys Lys Ile Lys Gly Val Ile Val Met AspMet Val 420 425 430 Asp Tyr Ile Ser Thr Asn Ile Leu Lys Tyr His Lys GlnLeu Tyr Asp 435 440 445 Lys Met Ser Thr Phe Glu Tyr Lys Arg Asp Ile LysSer Cys Lys Cys 450 455 460 Ser Ile Cys Ser Asp Ser Ile Thr His His IleTyr Glu Thr Thr Ser 465 470 475 480 Cys Ile Asn Tyr Lys Ser Thr Asp AsnAsp Leu Met Ile Val Leu Phe 485 490 495 Asn Leu Thr Arg Tyr Leu Met HisGly Met Ile His Pro Asn Leu Ile 500 505 510 Ser Val Lys Gly Trp Gly ProLeu Ile Gly Leu Leu Thr Gly Asp Ile 515 520 525 Gly Ile Asn Leu Lys LeuTyr Ser Thr Met Asn Ile Asn Gly Leu Arg 530 535 540 Tyr Gly Asp Ile ThrLeu Ser Ser Tyr Asp Met Ser Asn Lys Leu Val 545 550 555 560 Ser Ile IleAsn Thr Pro Ile Tyr Glu Leu Ile Pro Phe Thr Thr Cys 565 570 575 Cys SerLeu Asn Glu Tyr Tyr Ser Lys Ile Val Ile Leu Ile Asn Val 580 585 590 IleLeu Glu Tyr Met Ile Ser Ile Ile Leu Tyr Arg Ile Leu Ile Val 595 600 605Lys Arg Phe Asn Asn Ile Lys Glu Phe Ile Ser Lys Val Val Asn Thr 610 615620 Val Leu Glu Ser Ser Gly Ile Tyr Phe Cys Gln Met Arg Val His Glu 625630 635 640 Gln Ile Glu Leu Glu Ile Asp Glu Leu Ile Ile Asn Gly Ser MetPro 645 650 655 Val Gln Leu Met His Leu Leu Leu Lys Val Ala Thr Ile IleLeu Glu 660 665 670 Glu Ile Lys Glu Ile 675 64 amino acids amino acidlinear protein not provided 194 Lys Leu Tyr Thr Ile Ile Asp Thr Leu ProCys Pro Thr Cys Arg Ile 1 5 10 15 His Ala Lys Glu Glu Leu Thr Lys HisAsn Ile Met Ser Ser Asn Asp 20 25 30 Ile Asn Tyr Ile Tyr Tyr Phe Phe IleArg Leu Phe Asn Asn Leu Ala 35 40 45 Ser Asp Pro Lys Tyr Lys Ile Gln LeuAsp Lys Val Ala Pro Leu Val 50 55 60 583 base pairs nucleic acid doublelinear DNA (genomic) NO NO Swinepox virus Kasza S-SPV-001 CDS 2..583 195A AGC TTA AGA AAG AAT GTA GGG AAC GAA GAA TAT AGA ACC AAA GAT 46 Ser LeuArg Lys Asn Val Gly Asn Glu Glu Tyr Arg Thr Lys Asp 1 5 10 15 TTA TTTACT GCA TTA TGG GTA CCT GAT TTA TTT ATG GAA CGC GTA GAA 94 Leu Phe ThrAla Leu Trp Val Pro Asp Leu Phe Met Glu Arg Val Glu 20 25 30 AAA GAT GAAGAA TGG TCT CTA ATG TGT CCA TGC GAA TGT CCA GGA TTA 142 Lys Asp Glu GluTrp Ser Leu Met Cys Pro Cys Glu Cys Pro Gly Leu 35 40 45 TGC GAT GTA TGGGGG AAT GAT TTT AAC AAA TTA TAT ATA GAA TAC GAA 190 Cys Asp Val Trp GlyAsn Asp Phe Asn Lys Leu Tyr Ile Glu Tyr Glu 50 55 60 ACA AAG AAA AAA ATTAAA GCG ATC GCT AAA GCA AGA AGT TTA TGG AAA 238 Thr Lys Lys Lys Ile LysAla Ile Ala Lys Ala Arg Ser Leu Trp Lys 65 70 75 TCT ATT ATC GAG GCT CAAATA GAA CAA GGA ACG CCG TAT ATA CTA TAT 286 Ser Ile Ile Glu Ala Gln IleGlu Gln Gly Thr Pro Tyr Ile Leu Tyr 80 85 90 95 AAA GAT TCT TGT AAT AAAAAA TCC AAT CAA AGC AAT TTG GGA ACA ATT 334 Lys Asp Ser Cys Asn Lys LysSer Asn Gln Ser Asn Leu Gly Thr Ile 100 105 110 AGA TCG AGT AAT CTC TGTACA GAG ATT ATA CAA TTT AGT AAC GAG GAT 382 Arg Ser Ser Asn Leu Cys ThrGlu Ile Ile Gln Phe Ser Asn Glu Asp 115 120 125 GAA GTT GCT GTA TGT AATCTA GGA TCT ATT TCG TGG AGT AAA TTT GTT 430 Glu Val Ala Val Cys Asn LeuGly Ser Ile Ser Trp Ser Lys Phe Val 130 135 140 AAT AAT AAC GTA TTT ATGTTC GAC AAG TTG AGA ATA ATT ACG AAA ATA 478 Asn Asn Asn Val Phe Met PheAsp Lys Leu Arg Ile Ile Thr Lys Ile 145 150 155 CTA GTT AAA AAT CTA AATAAA ATA ATA GAT ATC AAT TAT TAT CCA GTG 526 Leu Val Lys Asn Leu Asn LysIle Ile Asp Ile Asn Tyr Tyr Pro Val 160 165 170 175 ATA GAA TCG TCT AGATCT AAT AAG AAA CAT AGA CCC ATA GGT ATC GGG 574 Ile Glu Ser Ser Arg SerAsn Lys Lys His Arg Pro Ile Gly Ile Gly 180 185 190 GTT CAG GGT 583 ValGln Gly 194 amino acids amino acid linear protein not provided 196 SerLeu Arg Lys Asn Val Gly Asn Glu Glu Tyr Arg Thr Lys Asp Leu 1 5 10 15Phe Thr Ala Leu Trp Val Pro Asp Leu Phe Met Glu Arg Val Glu Lys 20 25 30Asp Glu Glu Trp Ser Leu Met Cys Pro Cys Glu Cys Pro Gly Leu Cys 35 40 45Asp Val Trp Gly Asn Asp Phe Asn Lys Leu Tyr Ile Glu Tyr Glu Thr 50 55 60Lys Lys Lys Ile Lys Ala Ile Ala Lys Ala Arg Ser Leu Trp Lys Ser 65 70 7580 Ile Ile Glu Ala Gln Ile Glu Gln Gly Thr Pro Tyr Ile Leu Tyr Lys 85 9095 Asp Ser Cys Asn Lys Lys Ser Asn Gln Ser Asn Leu Gly Thr Ile Arg 100105 110 Ser Ser Asn Leu Cys Thr Glu Ile Ile Gln Phe Ser Asn Glu Asp Glu115 120 125 Val Ala Val Cys Asn Leu Gly Ser Ile Ser Trp Ser Lys Phe ValAsn 130 135 140 Asn Asn Val Phe Met Phe Asp Lys Leu Arg Ile Ile Thr LysIle Leu 145 150 155 160 Val Lys Asn Leu Asn Lys Ile Ile Asp Ile Asn TyrTyr Pro Val Ile 165 170 175 Glu Ser Ser Arg Ser Asn Lys Lys His Arg ProIle Gly Ile Gly Val 180 185 190 Gln Gly 51 base pairs nucleic aciddouble linear DNA (genomic) NO NO not provided 197 ACAGGAAACA GCTATGACCATGATTACGAA TTCGAGCTCG CCCGGGGATC T 51 138 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 198 GTATAGCGGC CGCCTGCAGGTCGACTCTAG ATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAATTGAATTGTAA TTATCGATAA TAAATGAATT CGATGGCTGT 120 GCCTGCAAGC CCACAGCA 138120 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 199 CTTAGCCCCA AACGCACCTC AGATCCATAA TTAATTAATT TTTATCCCGGCGCGCCTCGA 60 CTCTAGAATT TCATTTTGTT TTTTTCTATG CTATAAATGA ATTCGGATCCCGTCGTTTTA 120 116 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 200 GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGGTCTGGTGTCA AAAAGATCCA 60 TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTGCAGGCGGCCG CTATAC 116 51 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 201 TAATGTATCT ATAATGGTAT AAAGCTTGTATTCTATAGTG TCACCTAAAT C 51 51 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 202 ACAGGAAACA GCTATGACCA TGATTACGAATTCGAGCTCG CCCGGGGATC T 51 141 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 203 GTATAGCGGC CGCCTGCAGG TCGACTCTAGATTTTTTTTT TTTTTTTTTT TGGCATATAA 60 ATAGATCTGT ATCCTAAAAT TGAATTGTAATTATCGATAA TAAATGAATT CCATGTGCTG 120 CCTCACCCCT GTGCTGGCGC T 141 120base pairs nucleic acid double linear DNA (genomic) NO NO not provided204 TCGCCCGCCT CTGACGCCCC GGATCCATAA TTAATTAATT TTTATCCCGG CGCGCCTCGA 60CTCTAGAATT TCATTTTGTT TTTTTCTATG CTATAAATGA ATTCGGATCC CGTCGTTTTA 120116 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 205 GAAATCCAGC TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCAAAAAGATCCA 60 TAATTAATTA ACCCGGGTCG AGGCGCGCCG GGTCGACCTG CAGGCGGCCGCTATAC 116 51 base pairs nucleic acid double linear DNA (genomic) NO NOnot provided 206 TAATGTATCT ATAATGGTAT AAAGCTTGTA TTCTATAGTG TCACCTAAATC 51 45 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 207 CAAGGAATGG TGCATGCCCG TTCTTATCAA TAGTTTAGTC GAAAA 45 57base pairs nucleic acid double linear DNA (genomic) NO NO not provided208 TATATAAGCA CTTATTTTTG TTAGTATAAT AACACAATGC CAGATCCCGT CGTTTTA 57249 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 209 TCCAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAAGATCCATAAT 60 TAATTAACCA GCGGCCGCCT GCAGGTCGAC TCTAGATTTT TTTTTTTTTTTTTTTTGGCA 120 TATAAATAGA TCTGTATCCT AAAATTGAAT TGTAATTATC GATAATAAATGAATTCGGAT 180 CCATAATTAA TTAATTTTTA TCCCGGCGCG CCGGGTCGAC CTGCAGGCGGCCGCTGGGTC 240 GACAAAGAT 249 45 base pairs nucleic acid double linearDNA (genomic) NO NO not provided 210 CAAAAGTCGT AAATACTGTA CTAGAAGCTTGGCGTAATCA TGGTC 45 33 base pairs nucleic acid double linear DNA(genomic) NO NO not provided 211 CGACGGATCC GAGGTGCGTT TGGGGCTAAG TGC 3336 base pairs nucleic acid double linear DNA (genomic) NO NO notprovided 212 CCACGGATCC AGCACAACGC GAGTCCCACC ATGGCT 36 35 base pairsnucleic acid double linear DNA (genomic) NO NO not provided 213CCACGAATTC GATGGCTGTG CCTGCAAGCC CACAG 35 32 base pairs nucleic aciddouble linear DNA (genomic) NO NO not provided 214 CGAAGATCTG AGGTGCGTTTGGGGCTAAGT GC 32 34 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 215 CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG 34 32 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 216GAGCGGATCC TGCAGGAGGA GACACAGAGC TG 32 32 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 217 GCGCGAATTC CATGTGCTGCCTCACCCCTG TG 32 34 base pairs nucleic acid double linear DNA (genomic)NO NO not provided 218 CGCAGGATCC GGGGCGTCAG AGGCGGGCGA GGTG 34 32 basepairs nucleic acid double linear DNA (genomic) NO NO not provided 219GGGGAATTCA ATGCAACCCA CCGCGCCGCC CC 32 31 base pairs nucleic acid doublelinear DNA (genomic) NO NO not provided 220 GGGGATCCTA GGGCGCGCCCGCCGGCTCGC T 31

What is claimed is:
 1. A recombinant swinepox virus comprising a foreign DNA which (a) is inserted into a swinepox virus genome, wherein the foreign DNA is inserted within a region of the genome which corresponds to the 2.0 kb HindIII to BglII subfragment located within the HindIII M fragment of the swinepox virus genome and (b) is expressed in a host cell into which the virus is introduced.
 2. The recombinant swinepox virus of claim 1, wherein the foreign DNA is inserted within a BglII site within the region of the genome which corresponds to the 2.0 kb HindIII to BglII fragment.
 3. The recombinant swinepox virus of claim 1, wherein the foreign DNA is inserted into a NdeI site located within an open reading frame encoding swinepox virus thymidine kinase.
 4. The recombinant swinepox virus of claim 1, wherein the foreign DNA sequence encodes a polypeptide.
 5. The recombinant swinepox virus of claim 4, wherein the polypeptide is E. coli beta-galactosidase.
 6. The recombinant swinepox virus of claim 1, wherein the foreign DNA sequence encodes a cytokine.
 7. The recombinant swinepox virus of claim 6, wherein the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN).
 8. The recombinant swinepox virus of claim 6, wherein the cytokine is selected from a group consisting of interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors, and interleukin receptors.
 9. The recombinant swinepox virus of claim 4, wherein the polypeptide is selected from the group consisting of: human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicell-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, human immunodeficiency virus, rabies virus, measles virus, hepatitis B virus and hepatitis C virus.
 10. The recombinant swinepox virus of claim 4, wherein the polypeptide is hepatitis B virus core protein or hepatitis B virus surface protein.
 11. The recombinant swinepox virus of claim 4, wherein the polypeptide is equine influenza virus neuraminidase or equine influenza virus hemagglutinin.
 12. The recombinant swinepox virus of claim 4, wherein the polypeptide is selected from the group consisting of: equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Kentucky 92 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D.
 13. The recombinant swinepox virus of claim 4, wherein the polypeptide is selected from the group consisting of: hog cholera virus glycoprotein E2, hog cholera virus glycoprotein E2, swine influenza virus hemagglutinin, swine influenza virus neuraminidase, swine influenza virus matrix, swine influenza virus nucleoprotein, pseudorabies virus glycoprotein B, pseudorabies virus glycoprotein C, pseudorabies virus glycoprotein D, and PRRS virus ORF7.
 14. The recombinant swinepox virus of claim 4, wherein the polypeptide is selected from the group consisting of: Infectious bovine rhinotracheitis virus gE, bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase.
 15. The recombinant swinepox virus of claim 4, wherein the polypeptide is bovine viral diarrhea virus (BVDV) glycoprotein 48 or bovine viral diarrhea virus glycoprotein
 53. 16. The recombinant swinepox virus of claim 4, wherein the polypeptide is selected from the group consisting of: Marek's disease virus glycoprotein A, Marek's disease virus glycoprotein B, Marek's disease virus glycoprotein D, Newcastle disease virus hemagglutinin, Newcastle disease virus fusion protein, infectious laryngotacheitis virus glycoprotein B, infectious laryngotracheitis virus glycoprotein I, infectious laryngotracheitis virus glycoprotein D, infectious bronchitis virus glycoprotein VP2, infectious bronchitis virus glycoprotein VP3, infectious bronchitis virus glycoprotein VP4, infectious bursal disease virus spike protein, and infectious bursal disease virus matrix.
 17. The recombinant swinepox virus of claim 1, wherein the foreign DNA sequence is under the control of an endogenous upstream poxvirus promoter.
 18. The recombinant swinepox virus of claim 1, wherein the foreign DNA sequence is under the control of a heterologous upstream promoter.
 19. The recombinant swinepox virus of claim 18, wherein the promoter is selected from the group consisting of: pox synthetic late promoter 1, pox synthetic late promoter 2 early promoter 2, pox O1L promoter, pox I4L promoter, pox I3L promoter, pox I2L promoter, pox I1L promoter, pox E10R promoter, PRV gX promoter, HSV-1 alpha 4 promoter, and HCMV immediate early promoter.
 20. A homology vector for producing a recombinant swinepox virus by inserting foreign DNA into a nonessential site of a swinepox virus genome which comprises a double-stranded DNA molecule consisting of: a) double stranded foreign DNA not present within the swinepox virus genome; b) at one end the foreign DNA, double-stranded swinepox virus DNA homologous to the virus genome located at one side of the 2.0 kb HindIII to BglII subfragment located within the HindIII M fragment of the coding region of the swinepox virus genome; and c) at the other end of the foreign DNA, double-stranded swinepox virus DNA homologous to the virus genome located at the other side of the 2.0 kb HindIII to BglII subfragment located within the HindIII M fragment of the coding region of the swinepox virus genome.
 21. The homology vector of claim 20, wherein the foreign DNA sequence encodes a cytokine.
 22. The homology vector of claim 21, wherein the cytokine is chicken myelomonocytic growth factor (cMGF) or chicken interferon (cIFN).
 23. The homology vector of claim 21, wherein the cytokine is selected from a group consisting of: interleukin-2, interleukin-6, interleukin-12, interferons, granulocyte-macrophage colony stimulating factors, and interleukin receptors.
 24. The homology vector of claim 20, wherein the foreign DNA sequence encodes a polypeptide.
 25. The homology vector of claim 24, wherein the polypeptide is selected from a group consisting of: hog cholera virus gE1, hog cholera virus gE2, swine influenza virus hemagglutinin, swine influenza virus neuraminidase, swine influenza virus matrix protein, swine influenza virus nucleoprotein, pseudorabies virus gB, pseudorabies virus gC, pseudorabies virus gD, PRRS virus ORF7, and hepatitis B virus core protein.
 26. The homology vector of claim 24, wherein the polypeptide is selected from the group consisting of: equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Alaska 81 neuraminidase, equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D.
 27. The homology vector of claim 24, wherein the polypeptide is selected from the group consisting of: infectious bovine rhinotracheitis gE, bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, and the bovine parainf luenza virus type 3 hemagglutinin neuraminidase.
 28. The homology vector of claim 20, wherein the foreign DNA sequence is under the control of a promoter.
 29. A composition useful for inducing an immune response an animal against an animal pathogen which comprises an effective immunizing amount of the recombinant swinepox virus of claim 1 and a suitable carrier.
 30. A method of inducing an immune response in an animal against an animal pathogen which comprises administering to the animal an effective immunizing dose of the composition of claim
 29. 31. The recombinant swinepox virus of claim 4, wherein the polypeptide is derived from an organism selected from the group consisting of canine herpesvirus, canine distemper virus, canine adenovirus type 1, canine adenovirus type 2, parainfluenza virus, Leptospira canicola, icterohemorrhagiae parvovirus, coronavirus, Borrelia burgdorferi, Bordetella bronchiseptica, Dirofilaria immitis and rabies virus.
 32. The recombinant swinepox virus of claim 4, wherein the polypeptide is derived from a virus selected from the group consisting of feline leukemia virus, feline immunodeficiency virus, feline herpesvirus and feline infectious peritonitis virus. 